Modified-release morphine or placebo for chronic breathlessness: the MABEL trial protocol.

Chronic breathlessness, a persistent and disabling symptom despite optimal treatment of underlying causes, is a frightening symptom with serious and widespread impact on patients and their carers. Clinical guidelines support the use of morphine for the relief of chronic breathlessness in common long-term conditions, but questions remain around clinical effectiveness, safety and longer term (>7 days) administration. This trial will evaluate the effectiveness of low-dose oral modified-release morphine in chronic breathlessness. This is a multicentre, parallel group, double-blind, randomised, placebo-controlled trial. Participants (n=158) will be opioid-naïve with chronic breathlessness due to heart or lung disease, cancer or post-coronavirus disease 2019. Participants will be randomised 1:1 to 5 mg oral modified-release morphine/placebo twice daily and docusate/placebo 100 mg twice daily for 56 days. Non-responders at Day 7 will dose escalate to 10 mg morphine/placebo twice daily at Day 15. The primary end-point (Day 28) measure will be worst breathlessness severity (previous 24 h). Secondary outcome measures include worst cough, distress, pain, functional status, physical activity, quality of life, and early identification and management of morphine-related side-effects. At Day 56, participants may opt to take open-label, oral modified-release morphine as part of usual care and complete quarterly breathlessness and toxicity questionnaires. The study is powered to be able to reject the null hypothesis and an embedded normalisation process theory-informed qualitative substudy will explore the adoption of morphine as a first-line pharmacological treatment for chronic breathlessness in clinical practice if effective.

The Epstein-Barr Virus-Encoded EBNA1 Protein Activates the Bone Morphogenic Protein (BMP) Signalling Pathway to Promote Carcinoma Cell Migration.

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) protein is expressed in all virus-associated malignancies, where it performs an essential role in the maintenance, replication and transcription of the EBV genome. In recent years, it has become apparent that EBNA1 can also influence cellular gene transcription. Here, we demonstrate that EBNA1 is able to stimulate the expression of the Transforming growth factor-beta (TGFß) superfamily member, bone morphogenic protein 2 (BMP2), with consequential activation of the BMP signalling pathway in carcinoma cell lines. We show that BMP pathway activation is associated with an increase in the migratory capacity of carcinoma cells, an effect that can be ablated by the BMP antagonist, Noggin. Gene expression profiling of authentic EBV-positive nasopharyngeal carcinoma (NPC) tumours revealed the consistent presence of BMP ligands, established BMP pathway effectors and putative target genes, constituting a prominent BMP "signature" in this virus-associated cancer. Our findings show that EBNA1 is the major viral-encoded protein responsible for activating the BMP signalling pathway in carcinoma cells and supports a role for this pathway in promoting cell migration and possibly, metastatic spread.

Oral anticoagulation is preferable to injected, but only if it is safe and effective: An interview study of patient and carer experience of oral and injected anticoagulant therapy for cancer-associated thrombosis in the select-d trial.

BACKGROUND: Cancer patients have a four- to fivefold greater risk of thrombosis than the general population. Recommended treatment for cancer-associated thrombosis is 3-6 months of low-molecular-weight heparin. The 'select-d' trial is an open-label, randomised, multi-centre pilot trial in patients with cancer-associated thrombosis, utilising dalteparin (low-molecular-weight heparin) versus rivaroxaban (a direct oral anticoagulant), to assess effectiveness and safety. AIM: To explore patient and informal carers' experiences of cancer-associated thrombosis and their experience and understanding of the risk-benefit of thrombosis treatment. DESIGN: Qualitative substudy of the select-d trial, using semi-structured interviews. Interviews were audio-recorded and transcribed. Data were analysed using Framework Analysis. PARTICIPANTS: Participants were purposively sampled ( n = 37 patients; 46% male; age 40-89; 9 with carer present). RESULTS: Three themes were found: experience of cancer-associated thrombosis, experience of anticoagulation and risk-benefit balance of the two modes of administration. Some were shocked by their thrombosis diagnosis (most were unaware of their risk), but others found it insignificant compared with cancer. Most patients found tablets more convenient, but injections were acceptable in the context of having cancer. While most were happy to follow medical advice, others weighed preference on the basis of effectiveness. CONCLUSION: Lack of awareness of thrombosis risk is concerning; cancer patients must be informed to enable prompt help-seeking. Tablets could provide a welcome choice for patients if there is equivalent risk-benefit to injected anticoagulants. Patients trust their clinicians to tailor their treatment. Future research could explore the effect of routine information giving about the risk of thrombosis.

Signal Transduction Peptide of Tissue Factor Phosphorylated at Ser258 and the Unphosphorylated STP in Urine Are Potential Biomarkers for Bladder Cancer.

BACKGROUND: Procoagulant activity attributed to tissue factor (TF, CD142) bound to lipid microvesicles has previously been shown to be elevated in urine of patients with various solid cancers. The phosphorylation of the C-terminal signal transduction peptide (STP) at Ser253 and Ser258 has been determined to be important for the formation of TF-microvesicles. The purpose of this work was to investigate the marker potential of the TF-STP domain in urine of patients with cancer using immunologic methods to quantitate unphosphorylated TF and TF phosphorylated at Ser253 and Ser258. MATERIALS AND METHODS: We developed monoclonal and polyclonal antibodies directed against the 3 C-terminal STP species of TF and constructed 3 enzyme-linked immunosorbent assays (ELISAs) that specifically recognize unphosphorylated TF and TF phosphorylated at either Ser253 or Ser258. As proof of principle, a preliminary pilot study with stored Biobank-sourced urinary specimens from 45 healthy individuals and 38 patients with bladder cancer were studied using these ELISAs. RESULTS: We report that all 3 species of TF were found in the urine. Two species, TF-pSer258 and unphosphorylated TF, were significantly elevated in the cohort with bladder cancer. The sensitivity of TF-pSer258 by the receiver operator characteristic technique was 86.8%, with a specificity of 97.8% at a cutoff value of 0.55 ng/mL. Using a simplified sample preparation method for the ELISAs on the same clinical specimens, the sensitivity of TF-pSer258 was 86.8%, with a specificity of 93.3% at a cutoff value of 0.53 ng/mL. The unphosphorylated TF species was significantly elevated in later stage bladder cancer with best results seen for the unfractionated preparation technique (95% confidence interval, 10.55-15.74; N = 20) but not early stage non-muscle-invasive bladder cancer (95% confidence interval, 4.71-10.73; N = 18; P < .02). CONCLUSIONS: The development of these new ELISAs allows the quantitation of the urinary biomarkers TF-pSer258 and unphosphorylated TF, which may lead to a new diagnostic approach to the early detection of bladder cancer and warrant further investigation in a prospective trial.

Tumour and microparticle tissue factor expression and cancer thrombosis.

Cancer is frequently complicated by venous thromboembolic events (VTE), which pose a significant health burden due to the associated high morbidity and mortality rates, yet the exact details of the pathophysiological mechanisms underlying their development are yet to be fully elucidated. Tissue factor (TF), the primary initiator of coagulation, is often overexpressed in malignancy and as such is a prime candidate in predicting the hypercoagulable state. Further exploration of this potential role has identified increases in the number of TF-expressing microparticles (MP) in the circulation of cancer patients, in particular in those known to have high incidences of thromboembolic complications. The risk of VTE in cancer is found to be further elevated by chemotherapy. Chemotherapy may, in eliciting cancer cell apoptosis, result in an increase in release of circulating procoagulant MP. We discuss a potential role of elevated tumour TF expression and increased circulating TF-positive MP in predicting VTE risk.

Epstein-Barr virus-encoded EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma cells by inhibiting IKK phosphorylation.

BACKGROUND: The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB. RESULTS: In this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied. CONCLUSIONS: Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

Epstein-Barr virus-encoded EBNA1 modulates the AP-1 transcription factor pathway in nasopharyngeal carcinoma cells and enhances angiogenesis in vitro.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.