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Introduction:Ibrutinib (I), an irreversible Btk inhibitor, and venetoclax (V), a Bcl-2 inhibitor, improve CLL outcomes in trials compared to chemoimmunotherapy. I and V target two key pathophysiological pathways in CLL and should be synergistic. This is supported both by in vitrostudies and Phase II trials in which I+V results in high proportions of measurable residual disease (MRD) negativity. A Phase III trial comparing I+V (15 months [mo]) with chlorambucil-obinutuzumab led to the approval of I+V. However, mathematical disease modelling and Phase II studies favor defining duration of I+V according to individual patient sensitivity. We hypothesized that I+V is more effective than FCR in CLL and that treatment duration personalised using MRD response would optimize outcome.

Introduction and background

Introduction

In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph+ metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34+ cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph+ metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated β-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity.

Background

Heme-oxygenases (HOs) catalyze the conversion of heme into carbon monoxide and biliverdin. HO-1 is induced during hypoxia, ischemia/reperfusion, and inflammation, providing cytoprotection and inhibiting leukocyte migration to inflammatory sites. Although in vitro studies have suggested an additional role for HO-1 in angiogenesis, the relevance of this in vivo remains unknown. We investigated the involvement of HO-1 in angiogenesis in vitro and in vivo. Vascular endothelial growth factor (VEGF) induced prolonged HO-1 expression and activity in human endothelial cells and HO-1 inhibition abrogated VEGF-driven angiogenesis. Two murine models of angiogenesis were used: (1) angiogenesis initiated by addition of VEGF to Matrigel and (2) a lipopolysaccharide (LPS)–induced model of inflammatory angiogenesis in which angiogenesis is secondary to leukocyte invasion. Pharmacologic inhibition of HO-1 induced marked leukocytic infiltration that enhanced VEGF-induced angiogenesis. However, in the presence of an anti-CD18 monoclonal antibody (mAb) to block leukocyte migration, VEGF-induced angiogenesis was significantly inhibited by HO-1 antagonists. Furthermore, in the LPS-induced model of inflammatory angiogenesis, induction of HO-1 with cobalt protoporphyrin significantly inhibited leukocyte invasion into LPS-conditioned Matrigel and thus prevented the subsequent angiogenesis. We therefore propose that during chronic inflammation HO-1 has 2 roles: first, an anti-inflammatory action inhibiting leukocyte infiltration; and second, promotion of VEGF-driven noninflammatory angiogenesis that facilitates tissue repair.

Patients with secondary AML or MDS derived AML have poor outcomes compared to de-novo AML. The benefits of intensive chemotherapy without anticipated transplant consolidation have been previously doubted. Outcomes in USA trial centres have not often been closely replicable in real world settings. From November 2018 CPX-351 has been available in the UK for secondary AML, therapy related AML, AML with MDS related Karyotype (AML-MRC) and licensed but not funded for AML with myelodysplastic related changes.

Introduction:

Introduction:

Background

Both congenital and acquired antithrombin-III (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human alpha 1-antitrypsin (alpha 1AT) cDNA in yeast. Alteration of the alpha 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-III. Alteration of two further residues, to give a five-residue sequence identical to AT-III, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglycosylated and have shorter in vivo half-lives (t1/2) than human alpha 1AT. These antithrombins should be suitable for therapeutic replacement of AT-III in cases of congenital deficiency and in conditions associated with acquired AT-III deficiency, such as disseminated intravascular coagulation.

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 µmol/L) and the S558F (960 µmol/L) mutants compared with wild-type FVIII (>2,000 µmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 µmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.

The hemophilia A mutation database lists more than 160 missense mutations: each represents a molecular defect in the FVIII molecule, resulting in the X-linked bleeding disorder hemophilia A with a clinical presentation varying from mild to severe. Without a three-dimensional FVIII structure it is in most cases impossible to explain biological dysfunction in terms of the underlying molecular pathology. However, recently the crystal structure of the homologous human plasma copper-binding protein ceruloplasmin (hCp) has been solved, and the A domains of FVIII share approximately 34% sequence identity with hCp. This advance has enabled the building of a molecular model of the A domains of FVIII based on the sequence identity between the two proteins. The model allows exploration of predictions regarding the general features of the FVIII molecule, such as the binding-sites for factor IXa and activated protein C; it has also allowed the mapping of more than 30 selected mutations with known phenotype from the database, and the prediction of hypothetical links to dysfunction in all but a few cases. A computer-generated molecular model such as that reported here cannot substitute for a crystal structure. However, until such a structure for FVIII becomes available, the model represents a significant advance in modeling FVIII; it should prove a useful tool for exploiting the increasing amount of information in the hemophilia A mutation database, and for selecting appropriate targets for investigation of the structure-function relationships via mutagenesis and expression in vitro.

Both congenital and acquired antithrombin-lll (AT-III) deficiencies are amenable to replacement therapy. We describe two antithrombins produced by recombinant DNA techniques from human α 1-antitrypsin (a,AT) cDNA in yeast. Alteration of the α 1AT active site, replacing methionine 358 with arginine, results in a thrombin inhibition rate similar to that of heparin-activated AT-lll. Alteration of two further residues, to give a five-residue sequence identical to AT-lll, does not increase this rate further. Neither antithrombin is activated by heparin; both are unglyco-sylated and have shorter in vivo half-lives (t1/2) than human a, AT. These antithrombins should be suitable for therapeutic replacement of AT-lll in cases of congenital deficiency and in conditions associated with acquired AT-lll deficiency, such as disseminated intravascular coagulation.

Pemberton-Whiteley: CML Advocates Network: Membership on an entity's Board of Directors or advisory committees; Patient Evidence: Equity Ownership; AbbVie: Other: Grant funding; Amgen: Consultancy, Other: Grant funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Other: Grant funding, Speakers Bureau; Celgene: Consultancy, Other: Grant funding; Acute Leukemia Advocates Network (ALAN): Consultancy; Pfizer: Consultancy, Other: Grant Funding, Speakers Bureau; Takeda: Other: Grant funding; Shire: Other: Grant Funding; Kyowa Kirin: Other: Grant funding; Novartis: Consultancy, Other: Grant funding, Speakers Bureau; Incyte: Consultancy, Other: Grant funding; Jazz: Other: Grant funding, Speakers Bureau; Janssen: Consultancy, Other: Grant funding; Daiichi Sankyo: Other: Grant funding; Gilead: Other: Grant funding, Speakers Bureau; Leukaemia Care: Employment. Oliva:Celgene Corporation: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Apellis: Consultancy. Geissler:Incyte: Research Funding; Roche: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; UCB: Consultancy, Speakers Bureau; Servier: Consultancy; Takeda: Research Funding; Pfizer: Consultancy, Research Funding, Speakers Bureau; Biomarin: Consultancy; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding, Speakers Bureau. Wintrich:Takeda: Consultancy; Janssen: Other: Grant Funding; Celgene: Other: Grant Funding; Novartis: Consultancy, Other: Grant Funding. Salek:Pfizer: Honoraria, Speakers Bureau; Merck: Consultancy; Agios Pharmaceuticals, Inc.: Consultancy, Honoraria. Ionova:Takeda, BMS: Other: Principal Investigator of IISR, Research Funding. Tate:Quality Health: Employment. Bradley:Quality Health: Employment.

Sharf: Incyte: Honoraria, Other: Advocacy Advisory Board, Research Funding; Abbvie: Other: Advocacy grants funding; Roche: Other: Advocacy grants funding; BMS: Other: Advocacy grants funding, Research Funding; Takeda: Other: Advocacy grants funding; Janssen: Other: Advocacy grants funding; Novartis: Honoraria, Other: Advocacy Advisory Board, Research Funding; Pfizer: Honoraria, Other: Advocacy Advisory Board, Research Funding. Hochhaus:Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; MSD: Research Funding; Incyte: Research Funding. Geissler:Pfizer: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Research Funding, Speakers Bureau; Biomarin: Consultancy; Takeda: Research Funding; Amgen: Consultancy; Incyte: Research Funding; Roche: Consultancy; Servier: Consultancy; UCB: Consultancy, Speakers Bureau. Marin:Leukemia Patient Advocates Foundation: Employment. Pemberton-Whiteley:Acute Leukemia Advocates Network (ALAN): Consultancy; CML Advocates Network: Membership on an entity's Board of Directors or advisory committees; Patient Evidence: Equity Ownership; Daiichi Sankyo: Other: Grant funding; Gilead: Other: Grant funding, Speakers Bureau; Incyte: Consultancy, Other: Grant funding; Jazz: Other: Grant funding, Speakers Bureau; Janssen: Consultancy, Other: Grant funding; Kyowa Kirin: Other: Grant funding; Novartis: Consultancy, Other: Grant funding, Speakers Bureau; Pfizer: Consultancy, Other: Grant Funding, Speakers Bureau; Takeda: Other: Grant funding; Shire: Other: Grant Funding; AbbVie: Other: Grant funding; Amgen: Consultancy, Other: Grant funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Other: Grant funding, Speakers Bureau; Celgene: Consultancy, Other: Grant funding; Leukaemia Care: Employment. Bombaci:CLL Advocate: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: grant funding ; CML Advocate: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Grant funding ; Incyte: Consultancy; Onlus: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: grant funding; Novartis Pharma AG: Consultancy, Other: Advisory board ; MPN Advocate: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: grant funding ; Abbive: Consultancy, Other: grading funding; Bristol- Myers: Consultancy, Other: Grant Funding; Jazz: Consultancy, Other: Grant funding ; janssen: Consultancy, Other: grant funding. Christensen:Novartis: Consultancy, Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board. Gouimi:AMAL: Membership on an entity's Board of Directors or advisory committees; Novartis Pharma: Consultancy, Other: Advisory Board; Bristol-Myers: Consultancy; Novartis: Consultancy, Other: funding grant; Takeda: Consultancy; Pfizer: Consultancy. Deekes:Novartis Pharma: Honoraria. Bradley:Quality Health: Employment.

Introduction:

Our data suggest that chronic phase CML patients destined to fail imatinib have more advanced disease than evident by morphological criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit, while sparing good-risk patients from unnecessary toxicity. The potential role of beta-catenin in the regulation of resistance genes suggests that targeting Wnt/beta-catenin signaling may be useful to overcome resistance.Druker: MolecularMD: Equity Ownership; Novartis Pharmaceuticals: ; Bristol-Myers Squibb: . O'Brien:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Wyeth: Research Funding. Deininger:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Calistoga: Research Funding; Genzyme: Research Funding.

Background: Patients with chronic phase CML who achieve a complete cytogenetic response (CCR) have a low risk of disease progression. Since patients unlikely to achieve CCR may benefit from more aggressive therapy up-front, prediction of response prior to therapy would be useful. Our previous work has demonstrated that the gene expression profiles of unselected leukemia cells from prospective cytogenetic responders and non-responders are similar (Crossman et al. Haematologica90(4):459–64, 2005). Based on the hypothesis that cytogenetic refractoriness may be a property of leukemic progenitor rather than differentiated cells, we explored gene expression profiling of CD34+ cells as a tool for predicting CCR. Methods: Two independent data sets were generated. The first data set (learning set) was based on patients with CML who had either achieved a CCR within 1 year of imatinib therapy (R, n = 24), or remained at least 65% Ph+ (NR, n = 12). The prospectively collected, completely independent validation data set was based on 23 additional subjects using the same criteria (17 R and 6 NR). For the learning set CD34+ cells were isolated by FACS from bone marrow or peripheral mononuclear cells cryopreserved prior to imatinib therapy. For the validation set CD34+ were isolated from fresh cells using immunomagnetic columns. RNA was extracted and samples with ≥5ng of high quality total RNA were amplified and labelled with the Affymetrix two cycle cDNA synthesis and IVT labeling protocol using <20ng input RNA; 10μg of labelled target cRNA were hybridized to Affymetrix HG-U133 Plus 2.0 GeneChip® arrays. For both data sets, low-level analysis was done using Robust MultiArray Average (RMA) and gene-by-gene ANOVA was performed to determine differential expression between NR and R. Both statistical significance and effect size used to filter the ANOVA results. Parameters for the classification algorithms were chosen by nested cross-validation procedures. The classifier generated from the first study was then applied to the blinded validation set. Functional annotation clustering and over-representation analysis was also performed. Results: On the validation set, the classifier had an estimated accuracy rate of 85.7%. Based on transcript annotation analysis, there is significant over-representation of trancripts related to cell cycle, complement and coagulation, and apoptosis, among others. A functional cluster was identified related to regulation of transcription. Transcription factors up-regulated in cells from patients with subsequent cytogenetic refractoriness included ZNF168, MEIS1, KLF2, NFIB, MAF, FOSB and EGR1. In contrast, we found down-regulation of SERPINA1, THBD and PLAUR, a sub-network in the complement and coagulation cascades pathway that is associated with cell adhesion and migration. Conclusions: The transcriptional profiles of CD34+ cell from prospective cytogenetic responders and non-responders are distinct. A classifier based on the learning set predicted cytogenetic response in a totally independent validation set with ∼86% accuracy, making this one of the first prospectively validated classifiers. Examination of functional annotation for the transcripts in the classifier identified several functional clusters that are highly correlated with respect to direction of response (e.g. transcription factors) and may drive the biology of cytogenetic refractoriness.