James Sr Gibson

Changes in physiological, functional and structural markers of cystic fibrosis lung disease with treatment of a pulmonary exacerbation

Background Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures. Aim To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy. Methods A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers. Results Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=31), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=31), serum C-reactive protein (p<0.0001, n=34), serum interleukin-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=31). Discussion We identify the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with our study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.

An immunocytochemical assay to detect human CFTR expression following gene transfer

BACKGROUND To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery. METHODS We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR. RESULTS The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells. CONCLUSION We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.

P106 Inflammatory markers: data from the UK CF Gene Therapy Consortium Run-In Study

Inflammatory markers in sputum and serum have been used with variable success as outcome measures in interventional studies. Limited data are available on reproducibility of such assays in cystic fibrosis (CF) particularly over a long time period. This study was designed to address this; stable patients (FEV1>40, >10 years age) were recruited into the study which ran over an 18-month period with up to four hospital visits. Patients provided a 24 h sputum collection, for weighing at each visit. Spontaneous sputum was collected at the beginning of each visit; if insufficient sample was obtained, sputum was induced with hypertonic saline. Inflammatory markers were measured in dithiothreitol-processed sputum (total and differential cell counts, IL8 (and other cytokines), calprotectin, neutrophil elastase, myeloperoxidase and extracellular DNA). Blood was collected at each visit for cytokines (IL1β, IL6, IL8, IL10, IL12 (p40) and TNFα) and calprotectin. Data are available from 189 patients at 655 visits. Adequate sputum for analysis was obtained at only 60% of visits. Sputum induction accounted for only 16% of adequate samples. Median and range for each detectable assay are shown below. Serum cytokines IL1β, IL10, IL12 (p40) and TNFα were undetectable at each visit, and IL6 was only detectable in 17% of samples. To assess intra-individual variability the coefficient of variation of results across each visit for each patient is presented. Both sputum and serum assays showed a large range of results at each visit, but the variation for each individual was much higher than the ideal 10%. Serum assays were not able to discriminate between CF and non-CF, apart from calprotectin (CF 10.1(0.8–72.5) vs non-CF 0.40 (0.2–1.12) p<0.001). Due to the difficulty in obtaining sputum samples reliably and the large variability of results between visits in these stable patients, we consider it unlikely that a change due to a new therapy would be detectable. As such, we are not considering sputum inflammatory markers as primary or secondary efficacy endpoints in our multidose gene therapy trial. Serum markers also appear to be of limited use is assessing efficacy but will still be useful for toxicology and safety studies.Abstract P106 Table 1