Treatment of bleeding episodes in haemophilia A complicated by a factor VIII inhibitor in patients receiving Emicizumab. Interim guidance from UKHCDO Inhibitor Working Party and Executive Committee.

Emicizumab is a bispecific antibody that activates FX to FXa in the absence of FVIII. It has been shown to reduce bleeding episodes in people with haemophilia A complicated by a FVIII inhibitor. Despite the protection against bleeds, some breakthrough bleeds are inevitable and these may require additional haemostatic treatment. Emicizumab has been associated with severe adverse events when co-administered with activated prothrombin complex concentrate. To minimize the risk of adverse events, the UK Haemophilia Centre Doctors' Organisation issues the following updated interim guidance to its Inhibitor Guidelines for managing patients receiving Emicizumab based on the limit published information available in February 2018.

The incidence of factor VIII inhibitors in severe haemophilia A following a major switch from full-length to B-domain-deleted factor VIII: a prospective cohort comparison.

Although it has been suggested that switching of factor VIII (FVIII) products may increase inhibitor formation this is disputed. Half of UK patients changed rFVIII brands because of national contracting in 2010, presenting an opportunity to compare inhibitor incidence of switchers with non-switchers. Centres were requested to test all the patients for inhibitors prior to the switching date and 6-monthly thereafter. Positive and negative inhibitor test data were also collected to analyse for testing bias. A total of 1198 patients with severe haemophilia A and treated with Advate, Kogenate/Helixate or Refacto AF preswitch were included in the analysis, of whom 516 switched to Refacto-AF and 682 did not switch products. Five new inhibitors were reported amongst previously treated patients (>50 exposure days) with a median titre at the time of detection of 1.25 BU mL(-1) (IQR 0.7-23.05). One inhibitor occurred in a non-switcher using Kogenate, an incidence of 1.5 per 1000 treatment-years (95% CI 0.2-10.5). Four inhibitors arose in patients who had switched from Kogenate (two) or Advate (two) to ReFacto-AF, an incidence of 7.8 per 1000 treatment-years (95% CI 2.9-20.8). These incidence rates did not differ significantly from one another (incidence rate ratio 5.3 (95% CI 0.5-260.3) or from the historical rate of 6.05 inhibitors/1000 treatment-years (95% CI 5.18-7.06). Only one inhibitor (non-switcher) persisted. Non-switchers were significantly older (P = 0.03), and used significantly less FVIII per year (P = 0.005) prior to switching. Following switching, factor usage increased similarly (P = 0.53) in both groups. Switching from FLRFVIII to Refacto-AF (BDDRFVIII) was not associated with an increased inhibitor development.

The Genetics of bleeding disorders: a report on the UK Haemophilia Centre Doctors' Organisation annual scientific symposium, 10th October 2003.

The UK Haemophilia Centre Doctors' Organisation (UKHCDO) held its annual scientific symposium in October 2003, at the International Centre for Life, Newcastle-upon-Tyne. The educational day covered a range of topics relating to the genetics of bleeding disorders, including advances in genetics and gene therapy, antenatal diagnosis and counselling. We present the proceedings from the educational day.

Expression of angiogenic factors and hypoxia inducible factors HIF 1, HIF 2 and CA IX in non-Hodgkin's lymphoma.

AIMS: Angiogenesis in solid tumour pathology is well established but less is known about its role in haematological malignancies. Our study investigated the immunohistochemical expression of a variety of angiogenic and hypoxic factors and microvessel densities on 110 cases of high- and low-grade non-Hodgkin's lymphomas and reactive lymphoid tissues. methods and results: Expression of vascular endothelial growth factor (VEGF) was present in 82 (96%) of the non-Hodgkin's cases and 35 (100%) of the reactive lymphoid tissue cases. Both hypoxia inducible factors 1 alpha and 2 alpha (HIF 1 alpha, 2 alpha) were weakly expressed in the majority of high- and low-grade lymphomas. Carbonic anhydrase IX (CA IX), a HIF-inducible membrane-bound enzyme, expression was not abundant with membranous staining being present in seven (8%) of the lymphoma cases and none of the reactive cases. Thymidine phosphorylase (TP) was distributed amongst macrophages and follicular dendritic cells but was not present in the neoplastic population. The vasculature was stained using CD34 which gave rise to a distinct vascular, predominantly paracortical network present in low-grade lymphomas and reactive lymphoid tissue but which was lost in high-grade lymphomas. CONCLUSION: Our results suggest that non-Hodgkin's lymphomas may be less angiogenic and hypoxically driven than most solid tumours, which has implications for possible future therapies.

Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival.

Hypoxia inducible factors HIF1alpha and HIF2alpha are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1alpha and 2alpha protein levels, as a consequence of a redox-sensitive stabilization. The HIFalphas enter the nucleus, heterodimerize with the HIF1beta protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1alpha and HIF2alpha expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2alpha expression with HIF1alpha expression showed a significant association (P< 0.0001, r = 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF (P< 0.004), PD-ECGF (P< 0.003) and bFGF (P< 0.04) was noted. HIF1alpha correlated with the expression of bek-bFGF receptor expression (P = 0.01), while HIF2alpha was associated with intense VEGF/KDR-activated vascularization (P = 0.002). HIF2alpha protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD (P = 0.006). Analysis of overall survival showed that HIF2alpha expression was related to poor outcome (P = 0.008), even in the group of patients with low MVD (P = 0.009). HIF1alpha expression was marginally associated with poor prognosis (P = 0.08). In multivariate analysis HIF2alpha expression was an independent prognostic indicator (P = 0.006, t-ratio 2.7). We conclude that HIF1alpha and HIF2alpha overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting the HIF pathway may prove of importance in the treatment of NSCLC.

The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages.

The cellular response to hypoxia includes the hypoxia-inducible factor-1 (HIF-1)-induced transcription of genes involved in diverse processes such as glycolysis and angiogenesis. Induction of the HIF-regulated genes, as a consequence of the microenvironment or genetic changes, is known to have an important role in the growth of experimental tumors. Hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) are known to dimerize with the aryl hydrocarbon receptor nuclear translocator in mediating this response. Because regulation of the alpha chain protein level is a primary determinant of HIF activity, our aim was to investigate the distribution of HIF-1alpha and HIF-2alpha by immunohistochemistry in normal and pathological tissues using monoclonal antibodies (mAb). We raised a new mAb to detect HIF-1alpha, designated 122, and used our previously validated mAb 190b to HIF-2alpha. In the majority of solid tumors examined, including bladder, brain, breast, colon, ovarian, pancreatic, prostate, and renal carcinomas, nuclear expression of HIF-1alpha and -2alpha was observed in varying subsets of the tumor cells. HIF-2alpha was also strongly expressed by subsets of tumor-associated macrophages, sometimes in the absence of any tumor cell expression. Less frequently staining was observed in other stromal cells within the tumors and in normal tissue adjacent to tumor margins. In contrast, in normal tissue neither molecule was detectable except within subsets of bone marrow macrophages, where HIF-2alpha was strongly expressed.

Hypoxia-inducible expression of tumor-associated carbonic anhydrases.

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Relationship of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression to vascular endothelial growth factor induction and hypoxia survival in human breast cancer cell lines.

Hypoxia-inducible factors (HIF-1 and HIF-2) are two closely related protein complexes that activate transcription of target genes in response to hypoxia. Expression of HIF-1alpha and HIF-2alpha and their effects on survival under hypoxia were studied in six human breast cancer cell lines. We also evaluated the basal and inducible expression of two hypoxically regulated genes, vascular endothelial growth factor (VEGF) and lactate dehydrogenase-A (LDH-A). All of the cell lines studied expressed HIF-1alpha at various levels, but HIF-2alpha was low or absent from the more aggressive cell lines. There was an inverse correlation between HIF-1alpha and HIF-2alpha induction and clonogenic survival under hypoxia. Thus, cell lines with reduced induction of HIF-1alpha or HIF-2alpha showed high basal levels of VEGF and improved survival under hypoxia. A reduction in HIF expression was also associated with a more aggressive phenotype in vivo. To confirm these results, we carried out stable transfection of the MDA 435 cell line with human HIF-2alpha cDNA. There was no change in the growth rate in monolayer culture. However, in vitro growth as colonies and in vivo tumor growth of the HIF-2alpha overexpressing cells were significantly impaired compared with the control transfectants. Thus, despite the fact that HIF proteins are necessary for optimal tumor growth and angiogenesis in vivo, overexpression of these molecules seems detrimental to tumor growth. A balance between the angiogenic and tumor-inhibiting levels of HIF proteins may, therefore, be necessary for optimal tumor growth.

Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-1alpha.

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1alpha and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1alpha transactivation) of the VEGF promoter than the LDH-A promoter.