bluuebunny/crossref_metadata_2025 huggingface dataset:
GA Binding Protein (GABP) Is Required for Myeloid Cell Development and Differentiation.
Authors: Drumea Karen C. , Yang Zhong-fa , Rosmarin Alan G.
Published: April 2020

GABPα is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPα binds to DNA through its ets domain and forms a multimeric complex by recruiting its partner, GABPβ, which contains the transactivation domain. GABPα is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPβ to DNA. We cloned GABPα from a murine genomic BAC library and prepared a targeting vector in which the GABPα ets domain is flanked by loxP recombination sites (floxed allele, designated fl). Mice that bear one intact (and one disrupted copy) of GABPα, i.e. hemizygous mice, are phenotypically normal. Intercrossing of hemizygous mice yielded no nullizygous mice, indicating that homozygous loss of GABPα causes an embryonic lethal defect. To determine the effect of GABPα deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to mice that bear the interferon-responsive Mx1-Cre transgene, which express Cre in response to injection of the synthetic polynucleotide, poly I-C. Bone marrow cells underwent efficient deletion of GABPα following poly I-C injection; in contrast, other somatic tissues did not efficiently delete the floxed allele. Bone marrow, peripheral blood, and other tissues were examined for cellular morphology and flow cytometry. We compared mice that lack GABPα in bone marrow (i.e. fl/fl Mx1-Cre mice injected with poly I-C) to littermate controls (i.e. fl/fl mice injected with poly I-C). Mice that lack GABPα exhibited a striking and statistically significant decrease in granulocytes and monocytes in bone marrow and peripheral blood, compared with controls; in contrast, there was an increase in erythroid cells in GABPα null bone marrow. This indicates that the loss of GABPα has lineage-specific effects on myeloid cell development. Morphologic analysis indicates that mice which lack GABPα possess more immature granulocytes compared to control mice. Thus, GABP disruption causes a striking loss of myeloid cells in the bone marrow and peripheral blood of mice in a lineage-specific manner. Furthermore, the maturation block of murine granulocytes that is caused by GABPα disruption demonstrates the crucial role of GABP in myeloid differentiation.
Cyclin-Dependent Kinases Phosphorylate AML1 (RUNX1) and Regulate AML1 Transactivation.
Authors: Biggs Joseph R. , Peterson Luke F. , Zhang Youhong , Kraft Andrew S. , Zhang Dong-Er
Published: April 2020

The transcription factor AML1 (RUNX1) plays a critical role in normal hematopoiesis and in the development of leukemia. The phenotype generated by knockout of the AML1 gene demonstrates that AML1 is necessary for normal development of fetal hematopoietic cells. Conditional knockout of AML1 expression in adult bone marrow leads to lineage-specific effects on B- and T-cell maturation and pronounced inhibition of common lymphocyte progenitor production. In addition, the AML1-deficient adult mice exhibited inefficient platelet production and a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis composed of maturing myeloid and erythroid elements. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, can affect transcriptional activation. We have now shown that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (Cdks) Cdk1 and Cdk2. In addition, these two sites can be phosphorylated in vitro by purified, active Cdk1/cyclin B and Cdk2/cyclin A, but not by Cdk4/cyclin D. Mutation of AML1 serines 276 and 303 reduces AML1 activity during most phases of the cell cycle. In addition, mutation of serines 276 and 303 prevents phosphorylation from occurring at two nearby sites (serine 293 and threonine 300). Mutation of serine 293 and threonine 300 causes an increase of AML1 activity, especially in cells arrested in G2/M by treatment with nocodazole. Since Cdk phosphorylation can be stimulatory (serines 276 and 303) or inhibitory (serine 293, threonine 300), the interplay of Cdk phosphorylation at different sites might confer a very fine level of regulation of AML1 activity.
Gfi-1 Represses PU.1 Activity To Promote Granulocyte over Macrophage Differentiation.
Authors: Dahl Richard E. , Iyer Sangeeta R. , Cuylear Dorothy D. , Simon M. Celeste
Published: April 2020

Mice lacking the zinc finger transcriptional repressor protein Gfi-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Interestingly these immature Gfi-1−/− myeloid cells overexpress target genes of the PU.1 transcription factor: M-CSF receptor (M-CSFR) and PU.1 itself. Because of this we sought to determine if Gfi-1 modulates the transcriptional activity of PU.1. Here we demonstrate that Gfi-1 physically associates with PU.1 and represses PU.1- dependent transcription from the M-CSFR promoter. This interaction is functionally relevant as Gfi-1 blocks PU.1- dependent macrophage differentiation, but not granulocyte differentiation of a multipotential hematopoietic progenitor cell line. Additionally infection of primary bone marrow progenitors with a Gfi-1 retrovirus promotes granulocyte differentiation at the expense of macrophage differentiation. In contrast, a Gfi-1 mutant lacking repressor activity strongly promotes macrophage differentiation of hematopoietic progenitors. In transient transfections this Gfi-1 mutant not only blocks Gfi-1 repression of PU.1 transcriptional activity but C/EBP alpha mediated repression as well. From this data we hypothesize that Gfi-1 along with other transcriptional regulators target PU.1 for repression in the granulocyte-macrophage progenitor (GMP) in order to direct granulocyte differentiation. If true decreasing the concentration of PU.1 may decrease the need for Gfi-1 in directing granulocyte development. We are currently testing this in vivo by examining myeloid development in PU.1+/−Gfi-1−/− mice.
Transcriptional Regulation of the LMO2 T-Cell Leukaemia Oncogene.
Authors: Landry Josette-Renée , Kinston Sarah , Knezevic Kathy , Green Anthony R. , Göttgens Berthold
Published: April 2020

Transcriptional control has long been identified as a key mechanism regulating the formation and subsequent behaviour of haematopoietic stem cells. We have used a comparative genomics approach to identify transcriptional regulatory elements of the LMO2 gene, a transcriptional cofactor originally identified through its involvement in T-cell leukaemia and subsequently shown to be critical for the formation of haematopoietic stem cells and endothelial development. An initial stringent search for homology between evolutionary distant species demonstrated that, apart from the coding exons, high level of identity between mammalian, amphibian and fish sequences was restricted to the proximal promoter region of LMO2. Real-time RT-PCR expression analysis identified this promoter as the predominant source of transcription in haematopoietic tissue. Transient and stable transfections indicated that the proximal promoter was active in haematopoietic progenitor and endothelial cell lines and this activity was shown to depend on three conserved Ets sites which were bound in vivo by Elf1, Fli1 and Ets1. Transgenic analysis demonstrated that the LMO2 proximal promoter was sufficient for expression in endothelial cells in vivo. However, no haematopoietic expression was observed indicating that additional enhancers are required to mediate transcription from the proximal promoter in haematopoietic cells.

To identify additional elements involved in haematopoietic expression of LMO2, we have performed a less restrictive search for conserved sequences by comparing the human, dog, rat and mouse LMO2 loci to the marsupial opossum LMO2 locus. The addition of the opossum locus, and removal of the more distant fish and amphibian sequences from the alignment, resulted in the discovery of eleven conserved regions. These sequences represent candidate haematopoietic regulatory regions as they contain conserved transcription factor binding sites (E boxes, Ets and Gata sites) previously shown to regulate several other haematopoietic genes. We will present results from a systematic analysis of these regions for enhancer activity in both haematopoietic cell lines and transgenic mice, which suggest that several of these elements indeed act as enhancers. Taken together, our experiments will provide a framework for the transcriptional hierarchies within which LMO2 exerts its function in normal haematopoietic cells. Moreover, the current studies will serve as a platform to examine potential molecular mechanisms that can cause ectopic expression of LMO2 in T-cell progenitors with the ultimate consequence of developing T-ALL.
Increased Incidence of Metabolic Syndrome after Hematopoietic Stem Cell Transplantation.
Authors: Deliliers Giorgio Lambertenghi , Airaghi Lorena , Usardi Patrizia , Orsatti Alessandra , Baldini Marina , Giunta Valeria , Vener Claudia , Annaloro Claudio
Published: April 2020

Metabolic syndrome (MS), formerly known as X-syndrome, is a clinical and laboratory entity involving obesity, hypertension, and borderline fasting glucose, high triglyceride and low HDL cholesterol levels, that is considered to be a counterpart of insulin resistance with overweight playing a key role; increased insulin, leptin, adiponectin and resistin levels link its various features and some common flanking findings such as hypercholesterolemia, hyperuricemia and hyperferritinemia. Clinical and laboratory data reminiscent of metabolic syndrome are commonly observed after hematopoietic stem cell transplantation (HSCT). We evaluated the body mass index (BMI), routine lipid and glucidic markers, and common endocrinologic parameters in 37 HSCT recipients (19 males; median age 45 years, range 26–60; 22 autologous, 15 allogeneic) whose continuous CR lasted at least five years; insulin (baseline and after an oral glucose test), leptin, resistin, TNF-alpha and adiponectin levels were also detemined. Diabetic HSCT recipients were excluded. The control group consisted of 23 healthy volunteers of comparable age and sex without a family history of diabetes. In comparison with the controls, the HSCT recipients had significantly increased levels of TNF (median 10.05 vs 9.3 pg/mL, p=0.034), insulin (13.6 vs 10.1 mIU/mL, p=0.029) and leptin (13.71 vs 3.69 ng/mL, p= 0.02); there were no significant differences in adiponectin and resistin levels, BMI or the other considered parameters. In comparison with the other HSCT recipients, the 16 patients with higher baseline insulin levels (>15 mIU/mL) had significantly higher leptin levels (median 23.665 vs 8.3, p<0.001), and also different total cholesterol (231 vs 202 mg/dL, p<0.01), HDL-cholesterol (58 vs 68 mg/dL, p<0.01), triglyceride (150 vs 92 mg/dL, p<0.01) and ferritin levels (635 vs 491 ng/mL, p<0.01). This preliminary investigation show an increased prevalence of MS findings in HSCT recipients, and a relationship between baseline insulin levels and some common laboratory features of MS. Unlike in spontaneously occurring cases, obesity does not seem to play a primary role; furthermore, BMI and adiponectin were not significantly increased. However, the significant increase in TNF (another inducer of insulin resistance) suggests a possible pathogenetic sequence of post-HSCT MS linking TNF, insulin and leptin. The association between MS and the development of cardiovascular diseases is well known, but HSCT may, in alternative ways, induce a long-term metabolic derangement that affects the life expectancy of patients already cured of their baseline neoplastic disease.
Differences in Histone Acetylation Levels in CD34+ Cells between Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS): Correlation between Higher Levels of Histone 2B Acetylation and Survival in AML.
Authors: Gorre M. , Jilani I. , Chang R. , Bareng J. , Chan H. , Keating M. , Kantarjian H. , Estey E. , O’Brien S. , Giles F. , Albitar M.
Published: April 2020

Histone acetylation, along with DNA methylation, is a major epigenetic silencing mechanism believed to play a role in oncogenesis. Agents counteracting histone acetylation, such as the histone deacetylase (HDAC) inhibitors sodium phenylbutyrate and valproic acid, have shown promise in some types of leukemia and MDS. Despite the interest in HDAC inhibitors as therapeutic agents, little is known about the levels of histone acetylation and its clinical relevance in leukemia. In this study we used flow cytometry to quantify the levels of histone 2B acetylation (H2B-ac) and H3-ac in CD34+ and CD3+ cells in AML and MDS patients. Quantification using QuantiBRITE and PE (phycoerythrin)-labeled antibodies with a 1:1 ratio allowed us to specifically measure the antibody binding capacity in 100 CD34+ or CD3+ cells (molecules/100 CD34+ or CD3+ cells). While there was no significant difference in H2B-ac in CD3+ cells between previously untreated AML (50 patients) and previously untreated MDS (11 patients), a significantly higher percentage of CD34+ cells in MDS patients were positive for H2B-ac (P=0.03). In contrast, H3-ac levels were significantly higher in CD34+ and CD3+ cells in MDS than in AML (P=0.02 and P=0.03, respectively). This supports the concept that MDS is not simply a preleukemic disease and the blast population in MDS has characteristics different from those of blasts in AML. In patients with AML, higher levels of H2B-ac in CD34+ cells were associated with longer survival (P=0.03; Figure); multivariate analysis showed that this association was independent of cytogenetic abnormalities. This data confirms that epigenetic changes play a role in clinical behavior in patients with AML and that these patients may benefit from therapy that modifies histone deacetylation.

Figure Figure
Array CGH Discovers Novel Genomic Signatures in De Novo Acute Myeloid Leukemia (AML): Results of Children’s Oncology Group (COG) Study POG #9421.
Authors: Kuo Dennis J. , Lacayo Norman J. , Hoang Don , Juric Dejan , Raimondi Susana C. , Chen David , Chang Myron N. , Ravindranath Yaddanapudi , Weinstein Howard J. , Sikic Branimir I. , Dahl Gary Van Houten
Published: April 2020

Acute myeloid leukemia (AML) is a heterogeneous disease. Risk factors such as karyotype, FAB subtype, FLT3 status and response to induction therapy are determinants of outcome with current therapies. We hypothesize that array comparative genomic hybridization (CGH) will identify gene copy number changes that are determinants of outcome. Array CGH was performed on diagnostic bone marrow samples from patients on the COG study POG #9421. In order to determine regions of altered gene copy number, labeled genomic DNA samples were hybridized together with sex-matching normal human reference DNA to cDNA microarrays with 41,751 features (corresponding to 24,473 unique Unigene cluster IDs), arrays were obtained from the Stanford University Microarray Core Facility. Control hybridizations were performed to assess intra- and inter-experimental variability. We studied 70 samples with adequate high-quality DNA. Circular binary segmentation was used to distinguish discrete gene copy number transition points from chance noise events and to transform primary clone-by-clone data into genomic regions of equal copy number. Using gain/loss threshold, based on two-standard deviation range of control self-to-self distribution, novel gene amplifications and deletions were found in profiled samples. The highest alteration recurrence was observed for gains of chromosome 8 (21%) and losses of chromosome 6 (29%). The area of chromosome 8 which was found to be gained is notable for the presence of potential oncogenes such as ERK8. The deleted area of chromosome 6 is notable for the presence of potential regulators of oncogenesis: MDC1, DDR1, NFKBIL1, TNF, and BRD2. In summary, array CGH has identified novel areas of gene copy number gain and loss in this population of pediatric de novo AML patients. Further studies are needed to assess whether these genes are associated with outcome, known risk factors and whether they will provide insight into the heterogeneity of de novo AML.
Is Consolidation and/or Maintenance Treatment a Standard Therapy in Elderly AML in First Complete Remission?.
Authors: Corre Elise , Marie Jean-Pierre , Legrand Ollivier
Published: April 2020

The treatment of elderly patients with acute myeloid leukemia (AML) is very disappointing. Studies have established that if half of the intensivity treated patients achieve complete remission (CR), the high rate of relapse within the first year jeopardize the long term survival. No clearly effective postremission therapy had been established. Therefore we retrospective analyzed 141 elderly patients (>60 yo) in first CR, to evaluate the effectiveness of postremission therapy, consolidation (3+7or 2+5) and/or maintenance (low dose AraC). All patiens received a 3+7 induction therapy. In these 141 patients DFS and OS at 4 years are respectively 14%, and 19%.

According to the clinical status of the patient after induction, 20 (14%) patients did not receive therapy after induction, 30 (21%) patients received only maintenance therapy, 53 (38%) patients received only consolidation therapy, and 38 patients (27%) received both consolidation and maintenance therapy. These 4 groups were stratified according to age (< >70 yo) and WBC (< or > 30 x 109/l) (See Tables). The outcome of these patients receiving or not post induction therapy is shown in these Tables.

Patients DFS at 4 years OS at 4 years Consolidation No consolidation P value consolidation No consolidation P value Outcome of patients receiving or not consolidation therapy in 4 groups stratified according to age and WBC count < 70yo and WBC< 30x109/l 19% 13% p=0.01 27% 13% p=0.0074 <70yo and WBC>30x109/l 26% 0% p=0.08 38% 0% p=0.05 >70 yo and WBC < 30x109/l 0% 30% p=0.001 6% 30% p=0.01 >70 yo and WBC> 30x109/l 2% 0% NS 4% 0% NS

In patients > 70 yo and WBC < 30 x 109/l patients who received consolidation have a poorer prognosis than patients who did not because the mortality in first complete remission (mortality during consolidation) was high (25% versus 0% respectively, P=0.01).

Patients DFS at 4 years OS at 4 years Maintenance No Maintenance p value Maintenance No Maintenance P value Outcome of patients receiving or not maintenance therapy in 4 groups stratified according to age and WBC count <70yo and WBC<30x109/ 17% 20% NS 22% 25% NS <70 yo and WBC>30x109/l 23% 14% p=0.05 32% 23% NS >70 yo and WBC<30x109/l 25% 5% p=0.01 25% 5% p=0.008 >70 yo and WBC >30x109/l 2% 0% NS 4% 0% NS

In conclusion, in patients < 70 yo consolidation ± maintenance therapy (for DFS in patients with > 30 x109/l) improves the outcome of these patients (DFS and OS). In patients > 70 yo and with WBC < 30 x109/l maintenance therapy without consolidation, improves outcome. In these later patients (> 70 yo and with WBC < 30 x 109/l) consolidation therapy decrease outcome. In patients > 70yo and with WBC > 30 x 109/l, both consolidation and/or maintenance therapy does not improve outcome.
Circulating Endothelial Cells Are an Indicator of Response to Treatment in Acute Myeloid Leukemia Patients.
Authors: Wierzbowska Agnieszka , Robak Tadeusz , Krawczynska Anna , Wrzesien-Kus Agata , Pluta Agnieszka , Cebula Barbara , Smolewski Piotr
Published: April 2020

Introduction. The circulating endothelial cells (CEC) are proposed to be a noninvasive marker of angiogenesis. The number of CEC in peripheral blood of patients (pts) with acute myeloid leukemia (AML) has not been investigated so far.

Patients and Methods. We evaluated the count of resting (rCEC) and activated (aCEC) CEC and circulating endothelial progenitor cells (CEPC) as well as apoptotic CEC (CECAnnV+) in 62 AML pts at the time of diagnosis and 30 healthy controls. Additionally in 26 pts measurements were performed at the time of response evaluation and in 15 pts also 24 h after the first and last dose of chemotherapy. The levels of CEC were correlated with known prognostic factors and response to treatment. CEC were evaluated by the four colour flow cytometry using a panel of previously described monoclonal antibodies and an appropriate analysis gate. CEPC were defined as negative for hematopoietic marker CD45 and positive for endothelial cells markers CD34, CD31 and the endothelial progenitor marker CD133. Resting CEC were defined as CD45−, CD133−, CD31+, CD34+, CD146+ and negative for activation markers (CD105, CD106). CD105 or CD106 positive mature endothelial cells were classified as activated CEC. Apoptotic CEC were CD146 and Annexin V positive.

Results. In untreated AML pts we observed 10-fold higher CEC level (median 29,3/μL) than in the control group (2,95/μL) p<0,0001. The numbers of aCEC (12,7/μL), rCEC (12,3/μL) and CEPC (1,7/μL) were significantly higher in AML pts at diagnosis when compared to healthy controls (aCEC 0,9/μL, rCEC 1,6/μL and 0,1/μL; p<0,0001). CECAnnV+ count was also 10-fold higher in AML (1,5/μL) than in controls (0,15/μL; p<0,0001). Both CEC and CECAnnV+ counts did not correlate with WBC, hemoglobin and platelets count as well as percentage of blasts in bone marrow and absolute blast count. The positive correlations between CEC number and CEPC count (r=0,435; p<0,001), CECAnnV+ count (r=0,502; p<0,01) as well as LDH activity (r=0,328; p<0,02) were found. The significant decrease of aCEC and rCEC numbers 24 hours after the first dose of chemotherapy was noted in patients who achieved complete remission (CR)(p<0,04) but not in pts refractory to treatment. Moreover aCEC, rCEC, CEPC and CECAnnV+ counts determined at the time of response’s evaluation were significantly lower then at the time of diagnosis in pts who achieved CR (p<0,01) and did not differ in refractory AML. There was no difference between levels of both viable and apoptotic CEC in AML pts in CR and in the control group (p>0,05).

Conclusions. The CEC and CECAnnV+ levels are significantly higher in AML patients than in healthy subjects and correlate with response to treatment. Further investigation should be undertaken to better determine their prognostic and therapeutic value.
Tandem Duplications of the FLT3 and MLL Are Independent Poor Prognostic Factors in Pediatric Acute Myeloid Leukemia. AML99 Study in Japan.
Authors: Shimada Akira , Taki Tomohiko , Tabuchi Ken , Taketani Takeshi , Hanada Ryoji , Tawa Akio , Tsuchida Masahiro , Horibe Keizo , Tsukimoto Ichiro , Hayashi Yasuhide
Published: April 2020

To find poor prognostic factors other than karyotypes is desired to improve the treatment outcome in pediatric acute myeloid leukemia (AML). Total three hundread and eighteen patients were enrolled in Japanese Childhood AML Cooperative Treatment Protocol, AML99, from January 2000 to December 2002. We performed the mutation analysis of FLT3 and MLL in 158 patients (13 of 29 with FAB-M3, 10 of 49 with Down syndrome, 135 of 240 with other type of AML) Patients with FAB-M3 and patients with Down syndrome were treated on different protocol. Seventeen (12.6%) of 135 patients had FLT3-internal tandem duplication (ITD). The differences between AML patients with or without FLT3-ITD were significant in 4-year overall survival (OS) (35.3% vs. 86.1%, p<0.00001), disease free survival (DFS) (38.5% vs. 68.1%, p=0.0013), and relapse rate (RR) (53.8% vs. 28.5%, p=0.0029). Furthermore, MLL-partial tandem duplication (PTD) was found in 21 (15.6%) of 135 patients. The differences between patients with or without MLL-PTD were significant in 4y-OS (56.3% vs. 81.4%, p=0.013), DFS (41.7% vs. 68.3%, p=0.017), and RR (53.9% vs. 27.5%, p=0.0078). Out of 18 patients with MLL-PTD who received allo-SCT, 9 patients survived (6 of 8 in 1st CR, 3 of 4 in 2nd CR, 0 of 6 in non CR). Out of 12 patients with FLT3-ITD who received allo-SCT, 4 patients survived (4 of 6 in 1st CR, 0 of 6 in non CR). Allo-SCT is considered to be effective for the patients with TD of both genes. Coduplication of both genes was observed in 3 patients. FLT3 kinase domain mutation was found in 7.0% and MLL translocations were found in 11.4% of the patients, but both aberrations did not show poor outcome in this study. FLT3-ITD and MLL-PTD were found in 9 (25.7%) and 8 (22.9%) of 35 patients with normal karyotype. FLT3-ITD and MLL-PTD are considered to be independent poor prognostic factors of pediatric AML patients, especially in patients with normal karyotype. The different therapeutic approach including allo-SCT will be needed for these patients.
A Phase II Study of the Safety and Efficacy of a Novel Oral fXa Inhibitor (LY517717) for the Prevention of Venous Thromboembolism Following TKR or THR.
Authors: Agnelli Giancarlo , Haas Sylvia K. , Krueger Kathryn A. , Bedding Alun W. , Brandt John T.
Published: April 2020

Background: There is a continuing need for safe, effective and convenient therapies for prophylaxis of venous thromboembolism (VTE) after major orthopedic surgery. LY517717 (LY), an oral direct inhibitor of activated factor X (fXa), has been well tolerated in healthy subjects and is being developed to address these needs.

Objective: To assess the safety and efficacy of LY and to determine whether one or more doses of LY is non-inferior to enoxaparin in preventing VTE in patients undergoing total knee replacement (TKR) or total hip replacement (THR).

Methods: In a double-blind, double-dummy, dose-escalation study, patients undergoing TKR or THR were randomized to receive one of six oral doses of LY (25, 50, 75, 100, 125 or 150mg) or enoxaparin, 40 mg SC once daily. LY was administered fasting 6–8 hours after wound closure and then each morning. Enoxaparin was administered the night before surgery and then every evening. Both treatments were continued for a total of 6–10 doses. Patients underwent mandatory bilateral venography within 12 hours of the last dose of oral study drug and were assessed for symptomatic deep vein thrombosis (DVT), pulmonary embolism (PE) and bleeding events through day 30 (±7). All VTE and bleeding events were adjudicated by a blinded independent central adjudication committee. DVT detected by mandatory venography and symptomatic DVT and PE events were included in the combined VTE endpoint. Non-inferiority would be declared if the upper limit of the 90% confidence interval (CI) for the absolute difference in VTE incidence between an LY dose and enoxaparin was less than 0.14.

Results: 511 subjects were randomized. 507 patients received at least one dose of LY or enoxaparin and were included in the safety analyses. 391 patients had an evaluable bilateral venogram or experienced an objectively confirmed clinical VTE and were included in the efficacy analyses. The 3 lowest doses were stopped early due to lack of efficacy, and the study was completed with the 3 highest doses. VTE and bleed event data through the treatment and follow-up periods are summarized in the table. The 100, 125 and 150 mg doses of LY were non-inferior to enoxaparin in the incidence of symptomatic or venographic DVT or PE (upper limits for the 90% CI of the absolute differences are 0.13, 0.05 and 0.01, respectively). Adjudicated bleed events were uncommon in both LY and enoxaparin patients.

Conclusion: The 100, 125 and 150 mg doses of LY517717 were as efficacious as enoxaparin 40 mg for the prevention of VTE following TKR or THR in this study. LY was safe and well tolerated by study patients, with no significant difference in bleeding risk compared with enoxaparin.

Incidence of VTE and Bleed Events (%) (preliminary results)*
RHAMM/CD168-R3 Peptide Vaccination of HLA-A2+ Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) and Multiple Myeloma (MM).
Authors: Greiner Jochen , Giannopoulos Krzysztof , Li Li , Liebisch Peter , Wendl Christiane , Chen Jinfei , Ringhoffer Mark , Guillaume Phillipe , Ritter Gerd , Doehner Hartmut , Schmitt Michael
Published: April 2020

The receptor for hyaluronic acid mediated motility (RHAMM/CD168) has been described as a leukemia-associated antigen (LAA) eliciting both humoral and cellular immune responses in patients with hematological malignancies. RHAMM/CD168 is expressed in more than 80% of patients with acute myeloid leukemia (AML) or multiple myeloma (MM). Recently, we characterized the RHAMM/CD168-derived peptide R3 (ILSLELMKL) as a CD8+ T cell epitope. R3-primed CD8+ T lymphocytes were able to lyse autologous RHAMM/CD168+ AML blasts in a MHC class I-restricted and epitope-specific manner. Therefore, we initiated a phase I/II R3 peptide vaccination trial for patients with AML, MDS or MM overexpressing RHAMM/CD168. Patients were included with positive RHAMM/CD168 expression but with a limited tumor load. 300 mcg RHAMM R3 peptide emulsified with the incomplete Freund’s adjuvant (ISA-51, Montanide; day 3) and GM-CSF (Leukine, days 1–5) was administrated four times subcutaneously at a biweekly interval. The primary aim of the study is safety and feasibility of this peptide vaccination, secondary aims the evaluation of a specific T cell immune response to RHAMM/CD168 R3 peptide and the assessment of the influence of the R3 peptide vaccination on the remission status. Since January 2005, ten patients were enrolled in this study. The first eight patients (2 AML, 3 MDS, 3 MM) have completed the course of four vaccinations and four patients have been evaluated. The only side effects observed under R3-peptide vaccination were erythema and induration of the skin at the site of injection (CTC I°). In 2/4 patients, we found in the peripheral blood a significant increase of specific CD8+ T cells (from 0.01% to 0.8%) recognizing the R3 peptide in ELISPOT analysis and tetramer staining, one patient showed already initially a high number of HLA-A2/R3 tetramer+CCR7-CD27-CD45RA+ effector T cells and maintained this level of T cell response. Clinical responses have been assessed by the examination of peripheral blood and bone-marrow samples before and after vaccination. Patients showed a reduction of the tumor-specific expressed antigen RHAMM/CD168 in real-time RT-PCR analysis after vaccination. 2/4 patients with myeloid disorders (1 AML, 1 MDS RAEB1) showed a reduction of CD33+ cells in FACS analysis of the bone-marrow after 4 vaccinations from 10 and 7 % to 1–2 and <1%, respectively. One patient with MM showed a reduction of plasma cells in bone-marrow and a stable quantity of light chains in peripheral blood, one patient with AML showed a progressive disease. In summary, RHAMM/CD168 is a promising target antigen for immunotherapies in patients with hematological malignancies.
Gene Expression Profiling Predictive of Tipifarnib (ZARNESTRA®, R115777) Response in Patients with Newly Diagnosed Acute Myeloid Leukemia.
Authors: Raponi Mitch , Zhang Yi , Jatkoe Tim , Yu Jack , Lee Grace , Lancet Jeffrey E. , Karp Judith E. , Thibault Alain , Wang Yixin
Published: April 2020

The farnesyltransferase inhibitor (FTI), tipifarnib (ZARNESTRA®, R115777), has demonstrated clinical response in patients with hematological disease. While the inhibition of protein farnesylation is the primary mechanism of action (MOA), the level of farnesyl inhibition is not a reliable pharmacodynamic marker of response, nor is it clear what genetic markers can be employed to predict response. This prospectively designed study was conducted to identify potential genetic markers and expression signatures that may be surrogate predictors of response for tipifarnib in patients with acute myeloid leukemia (AML). Bone marrow samples were collected and gene expression profiles analyzed from a single arm phase 2 clinical study of tipifarnib in poor-risk patients with newly diagnosed AML (Lancet, et al. Blood. 2004;104:249a). In total, 79 samples were obtained before (n = 25), during (n = 30), and after (n = 24) tipifarnib treatment. Bone marrow samples were analyzed using the Affymetrix U133A GeneChip® array. Global gene expression signatures revealed that tipifarnib treatment resulted in gene expression changes that were maintained for up to 120 days following treatment termination. Pretreatment vs posttreatment samples identified approximately 500 genes that had significant changes (False Discovery Rate [FDR] <0.005) in gene expression following farnesyltransferase inhibition, including several genes associated with farnesylation (eg, K-ras, FNTA). Many of the modulated genes were identified as those significantly involved in protein biosynthesis, DNA replication, intracellular signaling, and cell cycle pathways, thus, reflective of inhibition of cellular proliferation. A subset of 27 genes (including genes associated with signal transduction and cell cycle) was also identified as being differentially modulated between responders and nonresponders (P <0.01). Gene expression signatures previously identified from a phase 2 clinical trial in relapsed and refractory AML were also tested in pretreatment samples to examine their ability to predict response (Raponi, et al. Blood. 2004;104:246a). A combination of 6 genes was found to have significant predictive accuracy in this independent set of samples (P = 0.05). The genes identified from these studies might be used as surrogate biomarkers of tipifarnib activity.
Use of a Disease-Specific Comorbidity Index To Describe Elderly Patients with Acute Myelogenous Leukemia (AML) and High Risk Myelodysplasia (MDS) Enrolled in a Phase II Study of CLORETAZINE™ (VNP40101M).
Authors: Giles Francis , Rizzieri David , Karp Judith , Vey Norbert , Khan Khuda , Mufti Ghulam , Hudak Diana , Karsten Verena , O’Brien Susan
Published: April 2020

Background: Elderly patients (pts) with AML generally have an unfavorable clinical course and are under-represented in clinical studies. Comorbid conditions are common in the elderly and, for older pts with AML, may have a major influence on choice of treatment. The ability to objectively measure comorbidity in this population may help predict patients’ risk for unacceptable toxicity. Comorbidity indices developed from a general medical population may underestimate the risks of cytotoxic treatment. An index developed specifically for pts with hematologic malignancies may be useful in categorizing pts by risk before selection of leukemia induction therapy. The Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI) is an index which captures comorbidities that predict non-relapse mortality (NRM) in pts with hematologic malignancies considered for allogeneic transplant (Sorror; Blood; 2005). The HCT-CI defines low risk with a score = 0, intermediate = 1–2 and high = ≥3. We used the HCT-CI to assess baseline comorbidity in an elderly pt population (median age 72) with untreated AML or high risk MDS enrolled in a single agent phase II study of CLORETAZINE.

Methods: Case report forms of 105 pts ≥ 60 years of age containing information about baseline medical history, concomitant medications, physical examination, and serum chemistries were reviewed. Using the HCT-CI definitions, pts were given scores representing the weighted sum of conditions that were active or required treatment at study entry.

Results: A total of 232 comorbid conditions were scored. In order of frequency, the most common were: cardiac (28%), including congestive heart failure, myocardial infarction, arrythmias, coronary artery stenosis requiring treatment, and valve disease; psychiatric disturbances (12%); hepatic dysfunction (11%); infection (11%); and ≥ grade 2 pulmonary disease (8%). Most pts (64%) had multiple comorbid conditions. Cardiac conditions were observed in 46% of all pts, psychiatric disturbances in 27%, hepatic disease in 25%, ongoing but controlled infections in 24%, and pulmonary disease in 18%. The response rate within each risk category is similar to the overall response rate, as summarized in the table below. The 19 early deaths (≤ 30 days) occurred in pts from the intermediate and high risk categories.

Risk Category N (%) # CR/CRp (%) # Early Deaths (%) Low (0) 11 (10) 4 (36) 0 Intermediate (1–2) 32 (30) 11 (34) 7 High (≥3) 62 (59) 18 (29) 12 Total 105 33 (31) 19 (18)

Conclusions: The HCT-CI is a helpful tool for defining comorbid conditions in elderly untreated AML pts. The majority of pts enrolled in the single agent Phase II CLORETAZINE study is at high risk for NRM as measured by HCT-CI score. Pts respond to CLORETAZINE across the HCT-CI scored risk groups.
Predictors of Clinical Response in Patients with High Risk Acute Myeloid Leukemia Receiving Treatment with the Histone Deacetylase Inhibitor Sodium Valproate.
Authors: Craddock Charles , Bradbury Charlotte , Narayanan Sujaatha , Rye Adam , Basu Supratik , MacWhannell Alan , Sawers Alistair , Cook Mark , Griffiths Michael , Grimwade David , Bunce Christopher , Turner Bryan
Published: April 2020

There is increasing evidence that perturbations in chromatin structure contribute to the pathogenesis of AML raising the possibility that modulators of chromatin structure may be of therapeutic benefit. Sodium valproate (VPA) is a potent histone deacetylase inhibitor (HDI) and in conjunction with ATRA induces apoptosis of primary AML blasts in vitro. It has been postulated that induction of pro-apoptotic genes including p21 and TRAIL play a critical role in determining the tumor selectivity of HDIs. We have therefore conducted a Phase I/II study of combined VPA/ATRA therapy in patients with high risk AML ineligible for chemotherapy and correlated clinical response with alterations in chromatin structure, induction of pro-apoptotic genes and in vitro sensitivity to VPA. In light of evidence that modulating cAMP levels may increase rexinoid responsiveness theophylline was added in patients who had failed to respond to 28 days combined VPA/ATRA therapy. A total of 20 patients (median age 72 yrs) have been treated to date including 12 patients with relapsed AML, 6 with previously untreated disease and 2 with primary refractory disease. Nine patients completed treatment for at least 8 weeks. Four patients (all with relapsed AML) demonstrated a haematological response. One patient achieved a complete remission and two a partial remission. Myeloblasts isolated from trial patients prior to the commencement of therapy demonstrated varying sensitivity to VPA and ATRA in vitro which correlated with clinical response. Combined treatment with VPA and ATRA resulted in increased levels of histone acetylation and methylation and induced expression of p15, p16 and p21 in myeloblasts from treated patients. Induction of TRAIL was documented in three patients-two of whom demonstrated a clinical response. p21 induction was documented in all responding patients. Induction of p21 and TRAIL was most marked in the patient who achieved a CR. There was no apparent correlation between induction of p15 or p16 and response. We have previously documented that exposure of AML cell lines to HDIs results in specific changes in HDAC expression patterns with selective up-regulation of HDAC11 and to a lesser extent HDAC9 and SIRT4. Similar changes in HDAC expression were documented in myeloblasts from patients after 28 days treatment with VPA/ATRA. This study demonstrates that VPA, in combination with ATRA and theophylline, has significant clinical and biological activity and identifies possible mechanisms underlying the tumor-specific activity of VPA in AML in vivo.
A Phase I Trial of CHIR-258, a Multitargeted RTK Inhibitor, in Acute Myeloid Leukemia (AML).
Authors: Morgan Gareth J. , Parker A. , Cavet J. , Cavenaugh J. , Heise C. , Garzon F.
Published: April 2020

Background: CHIR-258 is an orally active small molecule receptor tyrosine kinase (RTK) inhibitor which exhibits potent single digit nanomolar inhibitory activity against multiple RTKs involved in tumor growth and angiogenesis (IC50 <=10 for VEGFR, PDGFR, FGFR, C-KIT, and FLT-3). Approximately 30% of AML patients have a FLT-3 mutation (ITD-internal tandem duplication or catalytic domain) which drives the malignant process and confers a worse prognosis. Additionally, the inhibition of the angiogenesis component of the disease could be beneficial in all cases. Preclinical data have shown FLT3 ITD models to be ~25 fold more sensitive than FLT3 WT (Menezes, et al, Blood 2005). Our hypothesis was that AML blasts carrying FLT-3 mutations would be exquisitely sensitive to CHIR-258, but that there may also be activity against patients with wild type (wt) FLT-3.

Methods: CHIR-258 was administered orally to adult patients with relapsed/refractory AML on a 7 on/7 off schedule followed by continuous daily dosing for 28 days (1 cycle). Subsequent 28-day cycles of continuous dosing were permitted. Pharmacokinetic (PK) and pharmacodynamic (PD) assessments were serially performed and drug tolerability assessed.

Results: As of May 2005 8 patients have been treated in 2 (50mg and 100 mg) cohorts of 3–6 patients [median age: 62 years (range: 46–72), sex: 4M/4F, median prior relapses = 1 (range: 1–3), FAB subtypes: M1–1, M2–2, M5–1, M6–2, unclassifiable-1, no information-1] Five patients remain on study with a median number of treatment cycles=3 (range=1–11). There have been no DLTs to date. Dose escalation to 200 mg has been tolerated and accrual continues. Drug related adverse events include fatigue, anorexia, nausea/vomiting, and headache and were generally mild (CTC G1-2). Of the 8 patients treated, 1 had FLT-3 ITD mutation and 7 were wt for ITD and D835. A patient with AML-M6 with wt FLT-3 continues with stable disease after 11 cycles of therapy and was dose escalated from 50mg to 100mg after 8 cycles. The patient with FLT-3 ITD mutation treated at the 100mg dose had near complete clearing of blasts from marrow and peripheral blood. This patient later died due to a presumed fungal infection, not attributed to CHIR-258. Plasma exposure and Cmax increase proportionally across the dose range, and Cmax at the 100mg dose level is in the range where antitumor activity was seen in a preclinical oncogene addicted tumor models for FLT-3 ITD AML (MV4;11).

Conclusions: CHIR-258 has demonstrated activity in relapsed refractory patients with AML, both with a FLT-3 mutation and wt FLT-3. Accrual continues at the 200mg dose cohort and updated data will be presented at the meeting.
The Anti-Tumor Effects of SU11248, a Class III Receptor Tyrosine Kinase Inhibitor, Against a Variety of Human Hematological Malignancies.
Authors: Ikezoe Takayuki , Bandobashi Kentaro , Yang Yang , Saito Tsuyako , Togitani Kazuto , Tasaka Taizo , Koeffler H. Phillip , Taguchi Hirokuni
Published: April 2020

We studied anti-tumor effects of the class III receptor tyrosine kinase inhibitor SU11248 against a wide variety of hematological malignancies including the following leukemias: acute myeloid (THP-1, U937, Kasumi-1, MV4-11), acute lymphoblastic (NALL-1, Jurkat, BALL-2, PALL-1, -2), blast crisis of chronic myeloid (KU812, Kcl-22, K562) and adult T-cell (MT-1, -2, -4), as well as, non-Hodgkin’s lymphoma (KS-1, Dauji, Akata) and multiple myeloma (U266). Thymidine-uptake studies showed that SU11248 was active against MV4-11 and Kasumi-1 cells which possessed activating mutations of the FLT-3 and the c-Kit gene, respectively, with IC50s of approximately 30 nM. In addition, SU11248 inhibited the proliferation of Bcr/Abl-expressing KU812, Kcl-22, K562, PALL-1, and -2 cells with IC50s ranging from 2 to 5 uM. SU11248 induced apoptosis of these cells via activation of caspase 3 as measured by Annexin V staining and caspase assay. SU11248 inhibited phosphorylation of c-Abl and its downstream effectors Akt and STAT5 in these cells. Interestingly, mTOR inhibitor rapamycin enhanced the ability of SU11248 to inhibit the proliferation of Bcr/Abl-expressing cells. For example, SU11248 (2 uM, 48 h) or rapamycin (100 nM, 48 h) alone inhibited the proliferation of KU812 cells by 47 ± 6 % or 8 ± 6 %, respectively. When both were combined, their proliferation was inhibited by approximately 90 %. Furthermore, SU11248 (10 nM to 1 uM, 48 h) inhibited the proliferation of freshly-isolated leukemia cells from individuals with AML (15 cases) as well as Ph-positive ALL (1 case), who relapsed after treatment with conventional chemotherapy with imatinib. Profound growth inhibition by SU11248 (IC50s <10 nM) occurred in leukemia cells (4 cases) having an in-frame tandem duplications within the juxtamembrane region of FLT3 gene. Furthermore, rapamycin potentiated the anti-proliferative effects of SU11248 in the freshly-isolated leukemia cells. Taken together, SU11248 may be useful for treatment of individuals with variety of leukemias and the combination of SU11248 and rapamycin represents a promising novel treatment strategy.
Sequential Treatment with 5-Azacytidine, Valproic Acid (VPA), and All-Trans Retinoic Acid (ATRA) in Patients with High-Risk Acute Myeloid Leukemia (AML).
Authors: Raffoux Emmanuel , de Labarthe Adrienne , Gardin Claude , Daniel Marie-Therese , Chomienne Christine , Fenaux Pierre , Degos Laurent , Dombret Herve
Published: April 2020

Probably through inhibition of histone deacetylases, VPA ± ATRA has been associated with clinical responses in patients with myelodysplastic syndrome (MDS) or AML (Kuendgen, Blood 2004; Raffoux, Haematologica 2005). Interestingly, VPA/ATRA-induced differentiation of AML cells has been documented in vivo (Nervi, ASH 2004 #1805). On the other hand, the combination of VPA with a demethylating agent synergistically enhances its antileukemia activity in vitro (Yang, Leukemia Res 2005). In a pilot phase, we treated 5 high-risk AML patients with a sequential combination of 5-azacytidine, VPA, and ATRA. Each treatment cycle comprised 75 mg/m2/d 5-azacytidine SC from day 1 to 7, 20 mg/kg/d VPA (then adapted on serum concentration) from day 1 to 28, and 45 mg/m2/d ATRA from day 8 to 28. Six cycles were planned, each starting at day 29 of the previous one whatever the hematological status. High-risk AML (AML-M3 excluded) was defined as:

previously untreated de novo or post-MDS AML in patients aged 65+ unfit for intensive chemotherapy; or post-MDS AML in first relapse or AML in second or subsequent relapse.

Eligibility criteria also comprised WBC<30 G/L, PS<2, no severe infection, liver enzyme levels ≤ 2xULN, and serum creatinine ≤ 1.5xULN. Main patient characteristics and response are indicated in the Table below:

Patient Age AML Prior Tx BM blasts Cycles Response Time to response #1 71y post-MDS 4 41% 6 CRp 4 cycles #2 73y post-MDS 0 28% 6 CR 4 cycles #3 76y de novo 2 62% 2 Progression – #4 68y post-MDS 0 26% 2 Stable – #5 55y de novo 2 20% 4+ AML-free 3 cycles

Patient #3 progressed after the 2nd cycle. Treatment was also discontinued after the 2nd cycle in patient #4, because of ovarian carcinoma diagnosis. Response was observed after at least 3 cycles in the 3 remaining patients (1 CR, 1 CRp, 1 AML-free state with platelet count normalization), all treated as out-patients. Safety profile was excellent (no significant treatment-induced cytopenia). Main side effects were pain at 5-azacytidine injection sites and constipation. This treatment approach appearing promising in terms of efficacy versus toxicity, a formal multicenter Phase 2 study has been initiated.