bluuebunny/crossref_metadata_2025 huggingface dataset:
Severe Aortic and Mitral Valve Regurgitation Are Associated with von Willebrand Factor Defect.
Authors: Susen Sophie , Vincentelli Andre , Le Tourneau Thierry , Caron Claudine , Zawadzki Christophe , Prat Alain , Goudemand Jenny , Jude Brigitte
Published: April 2020

Severe aortic stenosis can be associated with mucosal and gastrointestinal bleeding probably related to loss of high molecular weight (HMW) multimers of VWF. Possible mechanisms of this loss are enhanced proteolysis by shear-dependant metalloprotease (ADAMTS 13) of HMW multimers of VWF or increased platelet-VWF interactions leading to clearance or degradation of HMW multimers of VWF. The aim of our study was to investigate wether this high shear-stress induced loss of HMW multimers of VWF is observed in other cardiac defects associated with high pressure blood flow such as severe aortic or mitral valve regurgitation.

Twenty four consecutive patients were operated on for aortic valve regurgitation (n=11) or mitral valve regurgitation (n=13). Before surgery recent mucosal bleeding was reported in 25% patients (n=5).VWF Closure Time of PFA 100® (CTADP) were prolonged in 87% of patients (n=20). The ratio of ristocetin cofactor activity to antigen and the ratio of collagen-binding activity to antigen were decreased in 28%(n=5) and 45%(n=10) of patients respectively. HMW multimers of VWF were decreased in 82 % (n=18) of patients. Six months after surgery, CTADP were normal in 71% (n=17) of patients, ratios were normal in all patients evaluable for this parameter (n=14) and HMW multimers of VWF increased in all patients as compared with preoperative value (8±2,5%vs10±2,3; p=0.04) but remain under value of control plasma in 63% (n=14) of patients.

In this study we demonstrated that VWF defect can be associated with severe aortic or mitral valve regurgitation and that platelet function-analyzer closure time, abnormalities of von Willebrand factor, or both could be frequent in those valve disease and might cause mucosal bleeding.
Relationship of Harvest CD34+ and CD3+ Counts To Chronic Graft-Versus-Host Disease (cGVHD), Relapse and Survival Following Sibling Allogeneic Stem Cell Transplant.
Authors: Kew Andrea K. , Couban Stephen , Thompson Kara , Djulbegovic Benjamin
Published: April 2020

G-CSF mobilized peripheral blood stem cells are increasingly used as a source of cells for sibling allogeneic transplantation. In comparison with bone marrow transplants (BMT), peripheral blood stem cell transplants (PBSCT) have been associated with increased chronic graft-versus-host disease (cGVHD). In order to examine the relationship between CD34+ and CD3+ counts and the development of cGVHD, we analyzed the outcome of patients from an international database of nine randomized trials comparing PBSCT and BMT. There were a total of 1111 patients: 305 patients developed extensive cGVHD, 261 limited cGVHD, 368 had no cGVHD and 177 did not have adequate information concerning cGVHD for analysis. We excluded patients with limited cGVHD because this can be a difficult diagnosis to make with certainty. We also excluded those without adequate data and thus the analysis is based on 673 patients (305 with extensive cGVHD and 368 with no cGVHD). There was no common baseline hazard for the development of extensive cGVHD among studies and thus the analysis was stratified by study. The primary outcomes were time to development of extensive cGVHD and time to the composite endpoint of death or relapse. Harvest CD34+ and CD3+ counts were analyzed as dichotomous variables based on the median cell counts. In the PBSCT group, the median CD34+ and CD3+ counts were 6.05 and 288.01×106/kg and in the BMT group, the median CD34+ and CD3+ counts were 2.89 and 32.79×106/kg respectively. As previously reported, there was an increased risk of developing extensive cGVHD with PBSCT compared to BMT (hazard ratio 1.77, 95% CI 1.39–2.23). The time to develop extensive cGVHD was not affected by the CD34+ or CD3+ count within either the PBSCT or BMT group. In addition, the composite endpoint of death or relapse was also not affected by either the CD34+ or CD3+ count in the PBSCT group. However, in the BMT group, a higher median CD3+ count was associated with an increased risk of death or relapse (p=0.04). Furthermore, a higher median CD34+ count was associated with a trend to a decreased risk of death or relapse (p=0.09). In summary, we confirm the higher risk of developing extensive cGVHD with PBSCT compared to BMT. In this analysis, we did not find a relationship between CD34+ and CD3+ counts and the risk of extensive cGVHD within either the PBSCT or BMT groups. However, our analysis suggests an association between higher median CD3+ counts and an increased risk of death or relapse in the BMT group. If this observation is confirmed, further studies are required to determine whether the higher CD3+ cell content of the marrow graft exerts a direct effect on the risk of relapse or death, or whether it is a surrogate for a less effective marrow harvest.
Pulmonary Complications after T-Cell Depleted Allogeneic Stem Cell Transplantation: Low Incidence and Strong Association with Acute GVHD.
Authors: Huisman Cynthia , van der Straaten Hanneke M. , Canninga-van Dijk Marijke R. , Fijnheer Rob , Verdonck Leo F.
Published: April 2020

Lung injury limits the success of hematopoietic stem cell transplantation (HSCT). The overall incidence varies from 30–50% and noninfectious causes occur in one third to one half of these. We reviewed pulmonary complications in 369 patients who received either allo-BMT or allo-PBSCT at our institution between 1993 and 2003. Control subjects were selected from the same database and matched on sex, underlying diagnosis, age, type of transplantation and cytomegalovirus-serostatus. For all patients the conditioning myeloablative regimen consisted of cyclophosphamide (60 mg/kg/day for 2 days) followed by total body irradiation (total lung dose 850 cGy). The graft was partially T-cell depleted (1–2 x 105 T cells/kg).

Sixty-one patients (16.5%) developed pulmonary complications, which were diagnosed at a median of 22 weeks after transplantation (range 2–263). Twenty-one patients (5.7%) developed infectious pneumonia. Non-infectious complications were further subclassified as BO (3.5%), BOOP (0.5%), DAH (0.8%), IPS (5.4%) or mixed etiology (0.5%). Acute GVHD ≥ grade II was significantly more common in patients with pulmonary complications than in the controls (36/61 versus 24/61 patients, P=0.02). There was no significant difference in the incidence of chronic GVHD (in 26/48 pulmonary patients versus 20/55 controls, P=0.1). Median survival was 41 weeks (range 4–583) for the pulmonary patients and 173 weeks (range 8–582) for the control subjects. These data illustrate that the incidence of pulmonary complications is low after T-cell depleted HSCT and demonstrate a clear association with acute GVHD. Improvement of the poor outcome of pulmonary complications is of utmost importance. Current studies at our institution are focused at the detection of early markers so that possible pre-emptive-like therapy can be initiated before symptomatic lung damage arises.
Effective Asparagine Depletion with Pegylated Asparaginase Results in Improved Outcomes in Adult Acute Lymphoblastic Leukemia (ALL) — Cancer and Leukemia Group B Study 9511.
Authors: Wetzler Meir , Sanford Ben L. , Owzar Kouros , Kurtzberg Joanne , DeOliveira D. , Frankel Stanley R. , Powell Bayard L. , Kolitz Jonathan E. , Bloomfield Clara D. , Larson Richard A.
Published: April 2020

CALGB study 9511 used pegylated asparaginase (PEG-ASP) in lieu of the native enzyme on days 5 and 22 of the 5-drug induction course and days 15 and 43 of the first intensification of the CALGB 8811 regimen (Blood1995; 85:2025). The aim of the study was to explore differences in the overall-survival (OS) and disease-free survival (DFS) profiles of those patients who did versus those who did not achieve asparagine depletion defined as enzyme levels > 0.03 units/ml for 14 consecutive days after at least one of four possible administrations of PEG-ASP (2000 U/m2, subcutaneously, capped at 3750 U). One hundred and four patients were enrolled between 7/95 and 12/97; 43F and 61M. Median age was 40 years (range, 17–71). Performance status (PS) was 0–1 in 81%. Thirty-one patients had an unfavorable [t(9;22), t(4;11), −7, +8] karyotype. Median WBC count was 86.5 x 109/L; 76/104 patients had > 30 x 109/L. PEG-ASP was tolerable (although grade 3 or 4 bilirubin occurred in 54%, hyperglycemia in 40%, and low fibrinogen in 30%). Treatment mortality was 9%. Antibodies to PEG-ASP were detected in 13/85 patients (15%). The complete remission (CR) rate was 76%. Median survival was 22 months [95% CI=13–29]. After a median follow-up of 90 months, 22 patients are alive without disease; three are alive after relapse. Samples were available from 85 eligible patients. Asparagine depletion was not significantly associated with achievement of CR (87.3% vs. 72.7%; P=0.55). Univariate analyses suggest that patients who achieved asparagine depletion (63 patients) had superior OS (two-sided P<0.0016; hazard ratio 2.4 [95%CI=1.4–4.2]) and DFS (P<0.011; hazard ratio 2.3 [95%CI=1.2–4.2]) profiles versus those who did not (22 patients). After adjusting, in the framework of an additive log-linear Cox model, for CR status (OS only), age, gender, PS, unfavorable karyotype, WBC count, detection of antibody to PEG-ASP, and transplantation, the difference between the OS profiles remained statistically significant (P<0.019; hazard ratio 2.1 [95%CI=1.1–3.9]) while the DFS profiles were borderline significant (P<0.052; hazard ratio 2.0 [95%CI=1.0–4.1]). We conclude that effective asparagine depletion with PEG-ASP as part of an intensive multi-agent therapeutic regimen in adult ALL is feasible and associated with improved outcomes.

Figure Figure
The Impact of Imatinib Therapy on Adult Philadelphia Positive Acute Lymphoblastic Leukaemia (ALL): Early Results from the UKALL12/ECOG 2993 Study.
Authors: Patel Bella , Goldstone Anthony H. , Ross Philip , Richards Sue , Foroni Letizia , Chopra R. , Fielding Adele
Published: April 2020

Modern protocols for the treatment of adults with Philadelphia positive (Ph+ve) ALL produce high complete remission (CR) rates. Despite this, the overall prognosis remains poor. Imatinib, an inhibitor of the BCR-ABL protein tyrosine kinase, may be a useful therapeutic agent for this disease. The arm of the MRC UKALL-XII/ECOG E2993 trial for patients with Ph+ve adult ALL was amended in 2003 to include Imatinib in the treatment protocol. Following induction chemotherapy those in haematological CR went on to receive treatment with Imatinib at 400mg/day, escalated to 600mg/day if tolerated. Those patients with a sibling, matched unrelated donor or related single haplotype match went on to allogeneic transplantation, whilst those with no available donor received an autograft. Chemotherapy alone was given to patients ineligible for a transplant procedure. All patients received Imatinib as maintenance therapy until frank relapse. 30 consecutive patients had a minimum of 12 months follow up. Sufficient data was available in 27 patients. The median age was 45 years (range 15 to 60), 59% were male, 96% were B-lineage, 4% were null lineage. Median white cell count at diagnosis was 24.1 X109/l. Median follow up was 15.6 months. The overall remission rate was 89%. The median time to CR was 28 days (range 19–93 days). Five (19%) patients received an autograft, 15 (56%) received an allograft. and 7 patients (26%) received chemotherapy alone. The event free survival (EFS) and overall survival (OS) at 1 year was 56% and 59% respectively. There were 7 deaths in remission (23%), 6 of which were transplant related. There were 7 relapses, 3 occurred in patients treated with chemotherapy alone, 2 prior to transplantation and 2 following allogeneic transplantation. These results were compared to 236 historic study patients in first CR after the same induction therapy but who did not receive Imatinib. There were no significant differences between the 2 groups with respect to gender, age, WBC or immunophenotype. Four percent received an autograft, 42% received an allograft and 47% received chemotherapy alone. A further 7% received a non-protocol transplant regimen. The EFS and OS of the historic controls was 35% and 56% respectively, with a comparable rate of deaths in remission (22%). Although the superior EFS in the Imatinib treated patients may be confounded by the greater number of allogeneic transplants in that group, the addition of Imatinib, even after achievement of CR does appear to enhance EFS in comparison to that seen in historical controls. Further follow up is needed to confirm an effect on overall survival and an independent anti-leukaemic effect of Imatinib in adult ALL.
Increased Stem Cell Mobilization Proficiency in Aged Mice.
Authors: Geiger Hartmut , Daria Deidre , Nattamai Kalpana J. , Xing Zhenlan
Published: April 2020

The ability of healthy, older individuals to undergo stem cell mobilization in response to granulocyte colony-stimulating factor (G-CSF) has not been specifically investigated. Aging is usually associated with an overall decline in tissue and cellular function. Therefore it is anticipated that the ability to mobilize stem cells in response to G-CSF may be reduced in aged individuals, although whether or not a negative correlation exists between age and mobilization is still under debate. We analyzed the influence of aging on the mobilization proficiency in healthy mice. Young 2–3 month old and aged 20–26 month old, which corresponds to a human age of 70, were mobilized according to standard procedures. The frequency of hematopoietic progenitor cells in peripheral blood (PB) of aged mice was 3- fold elevated over the frequency found in PB of young mice. The aged animals possessed the ability to mobilize increased numbers of primitive hematopoietic cells despite a slight, but significant anemia. This 3-fold difference in stem cell frequency in PB was further confirmed by long-term transplantation experiments. To determine if the increased ability to mobilize is intrinsic to aged hematopoietic stem cells or driven by age-associated, extrinsic changes in the BM microenvironment, a competitive mobilization assay based on the Ly5.2/Ly5.1 mismatch system was used. The genetic origin of the CFCs was determined by flow cytometry. In this assay, as previously reported, aged BM HSCs contribute to only 30% chimerism in PB prior to mobilization, resulting in a 1:2 ratio in favor of young cells in PB chimerism 3 months post-transplant. In contrast, when we analyzed the CFC chimerism of the competitively transplanted animals post-mobilization, 85% of all CFC from PB were derived from cells from old mice, strongly arguing that increased mobilization proficiency is a) not a simple consequence of the increased CFC content in aged BM and b) to a large extent intrinsic to the aged primitive hematopoietic cell, and thus relatively independent from the aged BM microenvironment. These data also imply that the stroma stem cell/interaction might change with age.
Genetic Interaction between SCL and c-Kit in Hematopoietic Cell Survival.
Authors: Lacombe Julie , Krosl Gorazd , Herblot Sabine , Martin Richard , Aplan Peter D. , Begley C.Glenn , Sauvageau Guy , Hoang Trang
Published: April 2020

The c-Kit tyrosine kinase receptor and the SCL/Tal1 (Stem Cell Leukemia) transcription factor are crucial to hematopoiesis and are co-expressed by hematopoietic progenitors. In the present study, we directly address the question whether SCL and c-Kit establish a code for the fate of multipotent progenitors in the adult. We therefore proceeded to loss and gain of function experiments, via retroviral mediated gene transfer in primary hematopoietic cells. We show that SCL is functionally required to sustain the survival of c-Kit+ cells in response to Steel Factor (Kit ligand), but not to GM-CSF and IL-3. Interfering with SCL function using a DNA-binding defective SCL mutant (ΔbSCL) caused a 100-fold loss of multipotent progenitors, due to massive apoptotic death in the c-Kit+ fraction, indicating that the DNA binding function of SCL is essential in cell survival. An antisense SCL also caused apoptosis in c-Kit+ cells, which was rescued by co-delivering SCL in the sense orientation. Furthermore, SCL sets threshold for the response of multipotent progenitors to Steel factor without affecting their response to IL-3 or GM-CSF, demonstrating the specificity of SCL for c-Kit-dependent pathway. Moreover, SCL levels in purified hematopoietic progenitors (HSC, CMP, MEP, CLP) were 3.5 to 15-fold decreased in W41 W41 mice that have a hypomorphic c-Kit allele when compared to age-matched wild type controls. Conversely, ectopic SCL expression is sufficient to bypass c-Kit signalling to suppress apoptosis and increase the proliferative potential of multipotent progenitors, as anemia and other hematopoietic defects caused by a hypomorphic c-Kit allele were rescued by introducing an SCL transgene into the W41 W41 background. We conclude that SCL operates within the c-Kit pathway to suppress apoptosis and determine the clonal output of adult multipotent progenitors. To define the pathway through which SCL suppresses apoptosis in hematopoietic progenitors, we performed microarrays analysis on Δb-SCL expressing cells and identified novel survival genes that were confirmed to be direct SCL targets by chromatin immunoprecipitation. Finally, the expression of these target genes was decreased in purified progenitors from W41 W41 mice as assessed by quantitative PCR. Together, this study reveals a new and essential genetic pathway involving SCL downstream of c-Kit signalling for the survival of hematopoietic progenitors.
p210BCR-ABL by down Regulating FLI-1 Reprograms Megakaryocytic Progenitors to the Erythroid Lineage.
Authors: Buet Dorothée , Raslova Hana , Juban Gaëtan , Geay Jean-François , Jarrier Peggy , Turhan Ali G. , Morle François , Vainchenker William , Louache Fawzia
Published: April 2020

Blood cells derive from a common multipotent stem cell that undergoes successive commitment steps with progressive restriction in differentiation ability. A close developmental relationship exists between the erythroid and megakaryocytic (MK) lineages as both lineages derive from a common bipotent progenitor. The nature of the events governing erythroid-specific or MK-specific programming is yet poorly understood. To approach this issue, we used an ectopic expression of the p210 BCR-ABL oncoprotein in adult primary human CD34+ progenitor cells. In culture conditions favoring the development of both erythroid and MK cells, we observed a major expansion of the erythroid lineage occurring at the expense of the MK lineage. The combined use of a p210BCR-ABL tyrosine kinase inhibitor together with single cell cloning experiments on prospectively isolated CD41+CD42− progenitor cells allowed us to identify that a part of MK progenitor cells were reprogrammed by p210BCR-ABL to take an erythroid fate. A similar lineage switching was seen after p210BCR-ABL expression in the human megakaryoblastic MO7e cell line. Microarrays and semi-quantitative RT-PCR studies showed that expression of p210BCR-ABL was associated with a decreased expression of the FLI-1 transcription factor. Moreover, re-expression of FLI-1 in p210BCR-ABL-transduced MO7e cells that had acquired an erythroid phenotype rescued the MK programming. Finally, a partial inhibition of FLI-1 in normal CD41+CD42− progenitor cells with a shRNAi led to an increased erythroid differentiation at the expense of MK differentiation. These results demonstrate that progenitor cells apparently committed to the MK lineage may be re-specified to the erythroid lineage by an ectopic expression of p210BCR-ABL and that the FLI-1 transcription factor is a key factor in the balance between erythroid versus MK commitment.
Protein Phosphatase2A Regulates Stromal Cell- Derived Factor-1 Mediated Responses of CD34+ Cord Blood Cells.
Authors: Basu Sunanda , Graham-Evans Barbara , Broxmeyer Hal E.
Published: April 2020

The chemokine stromal cell- derived factor-1(SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. During steady-state hematopoiesis, CXCR4/SDF-1 interaction restricts hematopoietic stem progenitor cells in marrow and disruption of the SDF-1/CXCR4 axis leads to their egress into circulation. However, biological responses to SDF-1 are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. It is known that phosphorylation of G-protein coupled receptors (GPCRs) by several protein kinases including serine-threonine kinase, protein kinase C (PKC), can result in receptor desensitization. This prompted us to investigate whether the role of serine- threonine phosphatase in the regulation of SDF-1 induced responses of CD34+ cord blood (CB) cells. To investigate the role of serine- threonine phosphatase we evaluated the effect of okadaic acid, a specific serine-threonine phosphatase inhibitor, on SDF-1 induced chemotaxis and adhesion of CD34+ cells from cord blood. Pre-incubation of CD34+ CB cells with okadaic acid (100–1000nM) significantly reduced SDF-1 directed chemotaxis and adhesion of CD34+ CB cells. This correlated with an increase in PKC phosphorylation. Although primary SDF-1 induced calcium flux in CD34+CB cells was unaffected by okadaic acid, if the cells were pretreated with SDF-1 and then stimulated with SDF-1, calcium flux was significantly less in CD34+CB cells pretreated with okadaic acid compared to untreated cells. Further, confocal microscopy was used to demonstrate the co-localization of serine threonine phosphatase and PKC. Using antibodies specific to PP2A catalytic subunit (PP2Ac) and various classes of PKC super family, it was observed in unstimulated CD34+ CB cells PP2Ac was mainly localized in nucleus and some phosphor-PKC-delta was detected in the cytoplasm. However, upon SDF-1 stimulation, co-localization of PP2Ac and phospho-PKC-delta was observed on the membrane of CD34+CB cells. These results indicate that serine-threonine phosphatase regulate SDF-1 mediated responses in CD34+CB cells and may play an important role in the ability of hematopoietic stem progenitor cells to continually respond to SDF-1.
LIF-LIF-R and SDF-1-CXCR4 Axes Regulate Overlapping and Complementary Steps of Metastasis of Rhabdomyosarcoma - Implication for Developing Better Antimetastatic Therapies.
Authors: Miekus Kasia , Wysoczynski Marcin , Reca Ryan , Jolanta Gozdzik , Salvatore Bertolone J. , Janowska-Wieczorek Anna , Ratajczak Mariusz Z.
Published: April 2020

Rhabdomyosarcoma (RMS) and skeletal muscle-derived tumors frequently infiltrate bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1-CXCR4 receptor (Blood2002;100:2597) and Leukemia Inhibitory Factor (LIF)-LIF- receptor (LIF-R) axes play a pivotal role in RMS metastasis to BM. In this study performed on primary patient material (n=23) and human alveolar and embryonal RMS cell lines, we compared various prometastatic effects of both axes. First, we found that both motomorphogens (LIF and SDF-1) and their corresponding receptors (LIF-R and CXCR4) are upregulated in hypoxic conditions. Second, both functional receptors were expressed on RMS cells; however, the expression of LIF-R in contrast to CXCR4 was PAX3-FKHR-independent and did not correlate with the alveolar rhabdomyosarcoma (ARMS) phenotype. Both ligands stimulated similar signaling pathways (MAPKp42/44 and AKT) in RMS cells and similarly stimulated directional chemotaxis. However, SDF-1 affected the spontaneous motility of RMS cells to a higher degree than LIF and strongly upregulated the secretion of MMPs and VEGF. In contrast, LIF enhanced the resistance of RMS cells to cytostatics (e.g., etoposide). To compare the biological effects of both axes we focused on the ARMS cell line RH30 (highly responsive to SDF-1 and LIF) and selected cells that responded to SDF-1 but not LIF (RH30-S) and to LIF but not SDF-1 (RH30-L) by using repetitive chemotaxis to SDF-1 or LIF. We found that RH30-L cells, as compared to RH30-S cells, responded worse in chemotactic assay to BM-derived conditioned media; however, they adhered much better to BM stroma. When both cell populations (RH30-S and RH30-L), together with the parental RH30 cells, were tested in vivo for their seeding potential in various organs in RAG2 immunodeficient mice, we found that RH30-L cells seeded better to BM, liver and lymph nodes than RH30-S cells. More important, the metastatic potential of RH30 cells was significantly reduced only when the inhibitors of both receptors, (T140 for LIF-R and gp130 blocking antibody for CXCR4) were employed together. Hence, since RMS cells respond to more than one chemoattractant, strategies aimed at targeting a single factor-receptor axis would be ineffective. Targeting of the LIF-LIF-R axis, along with the SDF-1-CXCR4 axis, could thus become a new, more effective antimetastatic strategy to inhibit RMS metastasis.
Sodium Nitrite Increases Regional Blood Flow in Patients with Sickle Cell Disease.
Authors: Mack A. Kyle , Machado Roberto F. , Sachdev Vandana , Gladwin Mark T. , Kato Gregory J.
Published: April 2020

Patients with sickle cell disease have decreased nitric oxide bioavailability, and studies from several groups have confirmed a blunted response to various NO donors in humans and mice with sickle cell disease. Recently published studies show that nitrite induces vasodilation in humans, apparently mediated by conversion of nitrite to NO.

This study is designed to determine the potential therapeutic effect of intra-arterial nitrite infusion to restore nitric oxide dependent blood flow in the forearms of patients with sickle cell disease.

Venous occlusion strain gauge plethysmography is used to measure the change of forearm blood flow in patients with sickle cell disease, before and after sequential brachial artery infusions of increasing doses of sodium nitrite. In addition, NO responsiveness before and after nitrite infusion is measured by test doses of the NO donor sodium nitroprusside (SNP). Six patients have completed the study and enrollment is continuing.

These data indicate that nitrite promotes regional blood flow in patients with sickle cell disease, albeit with a blunted response compared to our healthy control subjects, in whom we previously have found increased blood flow up to 187% with comparable dosing. The significant but blunted response is consistent with the state of nitric oxide resistance to NO donors that has been seen by several groups in patients and mice with SCD. Additionally, we find in these patients that nitrite partially restores SNP responsiveness, with baseline maximal SNP responses more than doubling on average following nitrite infusion, although this finding is preliminary. No adverse effects of nitrite were seen in these six patients.

Our early results support a role for nitrite as an NO donor effective in restoring NO-dependent blood flow in patients with sickle cell disease. Additional translational studies are warranted to evaluate the therapeutic effects of systemic nitrite dosing.

Table 1. Forearm Blood Flow Response to Nitrite Infusion Nitrite Dose (micromole/min) Sickle Cell Disease Historical Controls P< .0001 (ANOVA) 0.4 5 +/−7.2% N=6 22 +/−3.2% N=10 4 15 +/− 11% N=6 Not infused 40 49 +/− 8.9% N=6 187 +/− 16%N=18 Table 2. Nitrite Effect on Nitroprusside Responsiveness SNP Dose (micrograms/min) Pre-Nitrite Post-Nitrite P= .02 (RM-ANOVA) N=6 0.8 +21 +/− 5.6% +33 +/− 8.3% 1.6 +15 +/− 5.9% +62 +/− 15.1% 3.2 +29 +/− 6.3% +67 +/− 11.5%
Berkeley Sickle Mice Demonstrate CD8- and NK1.1-Dependent Increased Rejection of Allogeneic Hematopoietic Stem Cell Transplants.
Authors: Kean Leslie , Hamby Kelly , Perry Jennifer , Larsen Christian , Archerq David
Published: April 2020

While hematopoietic stem cell transplantation (HSCT) represents the only curative therapy for sickle cell disease, sickle patients undergoing HSCT face many complications, including an increased risk of graft rejection compared to non-sickle patients.

We have used the Berkeley sickle mouse model to study the potential mechanisms underlying this increased risk of rejection. Using a CD28/CD40 costimulation-blockade-based non-myeloablative HSCT regimen, we transplanted Berkeley sickle mice with fully allogeneic SJL bone marrow. While the vast majority (>85%, n=25) of control C57BL/6 animals became stably chimeric and immunologically donor-tolerant with this transplant regimen, sickle mice were much more prone to reject the transplant (~20% graft acceptance, n=25). Both CD8+ cells and NK1.1+ cells were found to contribute to this rejection, as depletion of either of these cell populations led to a marked increase in the percent of engrafted mice (>85% graft acceptance, n=15–25), while depletion of CD4+ cells led to the opposite effect, with 0% (n=25) animals engrafted in this depletion cohort.

The increased propensity of HSCT rejection in the Berkeley sickle mice may, in part, be explained by the presence of increased numbers of donor-reactive T cells (5–10-fold compared to C57BL/6 controls) in naïve sickle mice, despite their lack of exposure to donor antigens, and their housing in a Specific-Pathogen-Free environment. We speculate that these increased numbers of anti-donor T cells may occur as a result of heightened inflammation in the context of active sickle cell disease, which could lead to increased expansion and persistence of a T cell repertoire containing anti-donor heterologous T cell immunity. This heterologous immunity may have a profound effect on the success of HSCT for sickle cell disease, especially when non-myeloablative regimens are employed.
Electron Paramagnetic Resonance Study of Cell-Free Hemoglobin in Sickle Cell Disease: Potential Antioxidant Role of Haptoglobin.
Authors: Piknova Barbora , Gladwin Mark T. , Hillery Cheryl A. , Hogg Neil
Published: April 2020

It has previously been shown that red blood cell-free oxyhemoglobin (oxyHb) can affect endothelial function in Sickle Cell Disease (SCD) by virtue of its NO scavenging properties (Reiter et al, Nat Med, 2002). This reaction may have down-stream consequence via formation of metHb which has been demonstrated to have pro-oxidant properties and can oxidize low-density lipoprotein in vitro. In this study we have examined the fate of metHb in SCD vs normal plasma by examining the characteristic single line EPR signature of metHb in the g = 6 region. We show that the EPR signature of metHb differs in SCD compared with normals and exhibits a split EPR line indicative of a more rhombic heme geometry (See Figure).

Figure Figure

Addition of purified metHb to SCD plasma results in a conversion of the single line EPR spectrum to the rhombic form. By addition of hemin and hemoglobin to plasma constituents we have determined that the rhombic EPR spectrum is consistent with heme bound to serum albumin. In addition, immunoprecipitation of serum albumin from SCD plasma with EPR analysis of the IP fraction revealed the presence of albumin associated metHb. Interestingly, the transfer of heme from metHb to albumin is accelereated by the presence of low-density lipoprotein and inhibited by the addition of haptoglobin suggesting that plasma components can modulate heme release from metHb and that the lack of haptoglobin in SCD plasma allowed the transfer of heme from metHb to albumin. To confirm this we added metHb to SCD plasma in the presence and absence of haptoglobin and observed that the addition of haptoglobin prevented conversion of the metHb spectrum to the albumin associated split (rhombic) species. From these studies we conclude that plasma haptoglobin not only binds hemoglobin but also limits heme release from metHb. To examine potential oxidative consequences of this effect, we incubated LDL with metHb in the presence and absence of haptoglobin. Haptoglobin inhibited LDL oxidation in a dose-dependent manner. Interestingly, the 1-1 isoform of haptoglobin was much more effective than the 2-2 isoform, in agreement with published studies on the efficacy of hemoglobin binding to the two haptoglobin varieties. Additionally, we have measured levels of 8-isoprostanes in the plasma of SCD patients as an index of lipid oxidation and show that 8-isoprostane levels are significantly elevated in SCD patients (295 209 pg/ml, mean S.D., n= 13, vs. 69 37 pg/ml, mean S.D. n= 5 for normal controls). In conclusion, these studies provide evidence that the saturation of the haptoglobin clearance mechanisms in hemolytic disorders such as SCD can not only affect the NO-axis of endothelial function due to increases in cell-free hemoglobin, but can also lead to altered heme metabolism leading to lipid oxidation and formation of pro-inflammatory lipids which have the potential to alter both endothelial and red cell function.
Impact of Different Postremission Strategies in Younger Adults with Acute Myeloid Leukemia and Normal Karyotype Exhibiting a CEBPA Mutation.
Authors: Schlenk Richard F. , Corbacioglu Andrea , Fröhling Stefan , Du Juan , Späth Daniela , Schäfer Irina , Krauter Jürgen , Ganser Arnold , Döhner Hartmut , Döhner Konstanze ,   for the German-Austrian, AML Study Group (AMLSG)
Published: April 2020

Recently, mutations in the CEBPA gene, encoding CCAAT/enhancer binding protein alpha have been identified in 10 to 15% of patients with acute myeloid leukemia (AML) exhibiting a normal karyotype and mutant CEBPA was shown to predict favorable outcome. However, the impact of different postremission strategies such as high-dose cytarabine based chemotherapy (chemo), autologous (auto-SCT) or allogeneic stem cell transplantation (allo-SCT) in subgroups defined by these molecular markers is under discussion. Therefore, we initiated a pooled data analysis on patients exhibiting a normal karyotype at diagnosis treated within the prospective treatment trials AML-2/95, AML-1/99, AMLHD93 and AMLHD98A.

All patients (age 16–60 years) received two cycles of induction therapy with standard dose cytarabine combined with etoposide and idarubicin. After a first consolidation therapy, patients were assigned to an allo-SCT if an HLA-identical sibling donor was available in all four trials. In the AML-2/95 and AMLHD93 trials all other patients were assigned to chemo whereas in the AML-1/99 and AMLHD98A trials patients were randomised between auto-SCT and chemo. All patients were analyzed for CEBPA-mutations in the N-terminal transactivation domains and the C-terminal basic region-leucine zipper domain by direct sequencing of genomic DNA.

Between 1993 and 2004 a total of n=872 patients exhibiting a normal karyotype had been registered. Actually in 413 patients results of CEBPA analyses are available and 54 (13%) exhibited a mutation (46% N-terminal, 15% C-terminal, 39% N- and C-terminal). Response to induction therapy was 85% (46/54) and 74% (259/352) in the CEBPA mutated group and the CEBPA wild-type group, respectively. RFS and OS were 59% and 38% (p=0.01) as well as 66% and 41% (p=0.002) in the CEBPA mutated group and the CEBPA wild-type group, respectively after a median follow-up of 42 months. The distribution of postremission therapy of the 46 patients in the CEBPA mutated group was n=22 Chemo, n=13 allo-tpl, n=9 auto-tpl, n=2 allo-tpl from a matched unrelated donor. Analysis based on the availability of a matched related donor revealed a significant better RFS (p=0.03) and OS (p=0.03) for patients with an matched related donor.

Conclusion: Although AML patients with normal karyotype and a CEBPA mutation comprises a group with favorable prognosis allogeneic transplantation from an HLA-matched family donor seems to improve long term outcome. Confirmation of these preliminary data on a larger series of patients are necessary to improve a risk-adapted decision on postremission strategies.
Protein Kinase CK2 Is Associated with Poor Clinical Outcome and Regulates the Expression of Anti-Apoptotic Proteins and PTEN Phosphorylation in Acute Myeloid Leukemia.
Authors: Cheong June-Won , Eom Ju In , Lee Jin Koo , Park Sun Young , Kim Yuri , Park In Hae , Kim Jin Seok , Min Yoo-Hong
Published: April 2020

We previously demonstrated that the phosphorylation of c-terminal regulatory domain of phosphatase and tensin homologue (PTEN) protein are frequently observed and significantly associated with poor clinical outcome in acute myeloid leukemia (AML). Phosphorylation of PTEN stabilizes the PTEN protein, but makes it less active towards its substrate, phosphatidylinositol 3,4,5-triphosphate, resulting in an antagonizing the apoptotic pathways. Protein kinase CK2 is implicated in many cellular processes including the cell proliferation, survival, and tumorigenesis. However, the biological significance of CK2 expression and its association with PTEN phosphorylation has not been evaluated. In this study, we evaluated the protein kinase CK2 activity/expression and PTEN phosphorylation simultaneously in leukemic blasts obtained from 59 patients with AML with normal karyotype. Western blot analysis revealed that PTEN phosphorylation was observed in 30 (50.9%) cases. CK2 expression was significantly associated with CK2 catalytic activity and PTEN phosphorylation. Levels of CK2 catalytic activity were significantly higher in AML cases with PTEN phosphorylation (p-PTEN-positive) compared with those without PTEN phosphorylation (p-PTEN-negative) (P<0.05). Age, sex, white blood cell count, and complete remission rate were not different according to the CK2 expression. However, the overall survival was significantly shorter in AML cases with high CK2 expression (P< 0.05). Treatment of p-PTEN-positive AML cells with apigenin, a specific inhibitor of CK2, abolished the phosphorylation of PTEN, as well as Ser473 phosphorylation of Akt/PKB, in a dose-dependent manner. CK2 overexpression induced an increase in the proportion of cells in the G(2)/M phase. Induced overexpression of CK2 in U937 leukemia cells led to PTEN phosphorylation, Ser473 Akt/PKB phosphorylation, in addition to an increased expression of Bcl-2, Bcl-xL, Mcl-1, Survivin, and XIAP proteins. Taken together, these results suggest that the phosphorylation of PTEN and expression of anti-apoptotic proteins were directly regulated by protein kinase CK2 in AML cells. We also demonstrate, for the first time, that CK2 is an independent prognostic variable in AML patients with normal karyotype. These findings additionally extend our understanding of the role of CK2 in AML, and suggest the CK2 molecule as a potential target for AML treatment.
NPM Mutations in Adult AML with Normal Karyotype: A Retrospective Study of the Acute Leukemia French Association (ALFA).
Authors: Boissel Nicolas , Renneville Aline , Biggio Valeria , Philippe Nathalie , Thomas Xavier , Cayuela Jean-Michel , Terre Christine , Tigaud Isabelle , Castaigne Sylvie , Raffoux Emmanuel , de Botton Stephane , Fenaux Pierre , Dombret Hervé , Preudhomme Claude
Published: April 2020

NPM mutation (NPMm) has been recently reported as the most frequent mutation in AML, especially in the presence of a normal karyotype. Association of NPMm with an increased complete remission (CR) rate has been suggested, but the long-term prognosis is not known. The abnormal mutated NPM protein shows an aberrant cytoplasmic localization that allows an immunohistochemical detection of NPM status. However, all mutations reported are insertion/deletion of exon 12 resulting in a global insertion of 4 nucleotides. We therefore developed a NPMm detection test based on DNA PCR amplification and fragment length analysis. As previously reported, we observed a higher frequency of NPMm in AML with normal karyotype (47%, 50/106) than in CBF AML (0%, 0/7) or in AML with poor risk cytogenetic (13%, 4/32). We further evaluated the clinical profile and the prognosis of NPM mutations in a retrospective cohort of 106 patients with normal karyotype treated according to the ALFA-9000 or ALFA-9802 protocols between 1990 and 2004. In these patients aged 17–65 years, NPMm was significantly associated with a high white blood cell count (69 vs 18 G/L, p<.001) and an involvement of the monocytic lineage (FAB AML-M4/M5, p=.009). NPMm was also associated with a low rate of CEBPA mutation (CEBPAm, 10% vs 27%, p=.05) and a higher rate of FLT3 internal tandem duplication (FLT3/ITD, 38% vs 25%, p=NS). We did not find any correlation between NMPm and CR rate (86% vs 88%, p=NS). Long-term outcome did not differ between NPMm and NPM wild-type (NPMwt) patients neither in univariate analysis (6y-OS, 43% vs 37%; 6y-RFS, 47% vs 34%, p=NS) nor in multivariate analysis after adjustment on covariates significantly associated with prognosis in univariate analysis (age, CEBPAm, FLT3/ITD). NPM mutational status was available for 15 patients at relapse time. Ten patients out of 15 had NPM mutations at diagnosis and still displayed NPM mutations at relapse time. None of the 5 NPMwt patients acquired NPM mutation at relapse. Recently, we developed a RQ PCR technique to study minimal residual disease. Fifteen patients were evaluated at diagnosis and follow-up. In the majority of the cases a sensitivity of 10−4 was obtained. The high frequency, the stability, and the homogeneity of NPMm provide a promising minimal residual disease marker that we are prospectively evaluating. Prospective studies are also needed to confirm the definitive role of NPM mutation in the prognosis of AML patients.
FIP1L1-PDGFRA Positive Secondary Acute Myeloid Leukemia.
Authors: Metzgeroth Georgia , Popp Helena , Walz Christoph , Müller Martin C. , Mix Jürgen , Paschka Peter , Müller Lothar , Steder Martin , Del Valle Francisco , Aulitzky Walter E. , Benke Harro , Schoch Claudia , Haferlach Torsten , Hehlmann Rüdiger , Hochhaus Andreas , Reiter Andreas
Published: April 2020

The FIP1L1-PDGFRA positive myeloproliferative syndrome (MPS) represents a distinct clinicobiological entity with constitutively enhanced tyrosine kinase activity of the fusion protein and excellent response to treatment with imatinib. Most patients are initially diagnosed as idiopathic hypereosinophilic syndrome or chronic eosinophilic leukemia (CEL). A prominent clinical feature besides eosinophilia is the male predominance. However, the natural clinical course remains to be elucidated. Here we report on five male FIP1L1-PDGFRA positive patients (median age 58, range 46–64 years) with various subtypes of acute myeloid leukemia (AML) according to the FAB classification (M0, n=2; M2, n=1; M4eo, n=1; acute eosinophilic leukemia, n=1). All patients had a history of pronounced eosinophilia for a median interval of 6 mo (range 2–186) indicating the presence of a CML-like chronic phase disease evolving to blast crisis or secondary AML. One patient relapsed six years after conventional chemotherapy of a secondary FIP1L1-PDGFRA positive eosinophilia-associated AML M0 with typical features of CEL. Three patients had additional cytogenetic aberrations at diagnosis of AML similar to those frequently observed during clonal evolution of CML (trisomy 8, n=2; trisomy 9, n=1). All five patients received imatinib (100–300 mg/day) for a median time of 12 mo (range 1–23); three patients as monotherapy, one patient after one course and another patient after two courses of intensive chemotherapy. Three AML patients have been treated for more than 3 mo (12+, 18+, and 23+ mo) and are currently free of relapse. All three patients achieved a rapid complete hematological remission, defined as a complete clearance of blasts, normalization of peripheral blood and bone marrow eosinophil count, and a complete molecular remission 4, 5, and 14 mo, respectively, after start of treatment as determined by nested RT-PCR for the FIP1L1-PDGFRA transcript. In all cases treatment with imatinib was well tolerated. We conclude that the occurrence of the FIP1L1-PDGFRA fusion gene seems to be associated with a chronic phase disease which may progress to secondary AML. Therefore, all patients presenting with an eosinophilia-associated AML and normal karyotype should be screened for the cytogenetically invisible FIP1L1-PDGFRA fusion gene by RT-PCR and/or fluorescence in situ hybridization, in addition to the commoner CBFB-MYH11 fusion. Patients with a FIP1L1-PDGFRA positive MPS are excellent candidates for treatment with imatinib even if they present with secondary AML.
Prognosis Value of Flt3 Internal Tandem Duplication and P-Glycoprotein Functionality, in 171 AML.
Authors: Marzac Christophe , Teyssandier Irene , Perrot Jean Yves , Faussat Anne Marie , Tang Ruoping , Casadevall Nicole , Marie Jean Pierre , Legrand Ollivier
Published: April 2020

Adult acute myeloid leukemia (AML) with intermediate cytogenetic remains a very heterogeneous group of patients with highly variable individual prognosis. Emerging data suggest that some molecular markers could help to refine cytogenetic stratification. Here we focused on ABCB1 (MDR1/Pgp) ATP-binding cassette transporter activity and fms-like tyrosine kinase mutations (Flt3/ITD) because their individual prognosis value is well demonstrated and because they may lead to targeted therapy. Therefore we assessed Pgp activity using JC-1 probe and Flt3 exon 14 mutational status by standard PCR and realised a multivariate analysis in 171 adult AML patients treated in the EORTC protocols. Flt3/ITD (ITD+) and high Pgp activity (Pgp+) were found in 26/171 (15%) and 55/171 (32%) of the patients, respectively. The repartition was remarkable in that, as defined with our criteria, both were negative in 94/171 (55%, Pgp-ITD- group) or mutually exclusive in 73/171 (43%, Pgp-ITD+ and Pgp+ITD- groups) and only 4/171 (2%, Pgp+ITD+ group) were positive for both. In univariate analysis complete remission (CR) rates for ITD+ were 38% vs 61% for ITD- patients (p=0.034) and 47% vs 62% (p=0.06) for Pgp+ vs Pgp- patients. Individually, both parameters were strong predicators for overall survival (OS) (p=0,02) but not for disease free survival. In multivariate analysis Pgp+ITD+ (p<0.0001) and age (p=0.0022) were independent prognostic factors for CR achievement. For OS, CR achievement (p<0.0001), WHO performance status (p=0.0007) and Pgp+ITD+ (p=0.0014) were also independent prognostic factors. In 98 patients with intermediate cytogenetic CR rates for ITD+ were 40% vs 62% for ITD- patients (p=0.099) and 41% vs 67% (p=0.014) for Pgp+ vs Pgp- patients. In the Pgp-ITD- group (47/98 patients), CR rate was 70% vs 44% for others (p= 0.012). The 4 years OS achieved 48% vs 16% (p<0.0001) and DFS was 56% versus 27% (p=0.024). Furthermore, OS curves of the intermediate cytogenetic-Pgp-ITD- group were not significantly different from the favorable cytogenetic group. In conclusion Flt3-ITD and P-gp activity are independent and additive prognostic factors, which provide a powerful risk classification that can be used to stratify the treatment of intermediate cytogenetic AML patients.

Figure Figure