Maria H Dewar

Sputum Proteomics in Inflammatory and Suppurative Respiratory Diseases

RATIONALE Markers of inflammatory activity are important for assessment and management of many respiratory diseases. Markers that are currently unrecognized may be more valuable than those presently believed to be useful. OBJECTIVES To identify potential biomarkers of suppurative and inflammatory lung disease in induced sputum samples. METHODS Induced sputum was collected from 20 healthy control subjects, 24 patients with asthma, 24 with chronic obstructive pulmonary disease, 28 with cystic fibrosis (CF), and 19 with bronchiectasis. Twelve patients with CF had sputum sampled before and after antibiotic therapy for an infective exacerbation. The fluid phase of induced sputum was analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy on three protein array surfaces. Some protein markers were selected for identification, and relevant ELISA assays sought. For 12 patients with CF, both SELDI-TOF and ELISA monitored changes in inflammatory responses during infective exacerbations. MEASUREMENTS AND MAIN RESULTS SELDI-TOF identified potential biomarkers that differentiated each of the disease groups from healthy control subjects: at a significance of P < 0.01, there were 105 for asthma, 113 for chronic obstructive pulmonary disease, 381 for CF, and 377 for bronchiectasis. Peaks selected for protein identification yielded calgranulin A, calgranulin B, calgranulin C, Clara cell secretory protein, lysosyme c, proline rich salivary peptide, cystatin s, and hemoglobin alpha. On treatment of an infective CF exacerbation, SELDI-TOF determined falls in levels of calgranulin A and calgranulin B that were mirrored by ELISA-measured falls in calprotectin (heterodimer of calgranulins A and B). CONCLUSIONS Proteomic screening of sputum yields potential biomarkers of inflammation. The early development of a clinically relevant assay from such data is demonstrated.

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.

Moving from CFC Aerosol to HFA Aerosol or Dry Powder Inhalers: What Do Patients Think?

Background/Objectives: Environmentally friendly hydrofluoroalkane (HFA) pressurised metered-dose inhalers are currently being marketed to replace chlorofluorocarbon (CFC)-driven devices. It is uncertain whether these new formulations with different properties are acceptable to patients. Similarly, switching a patient to a dry powder inhaler (DPI) carries the risk of non-acceptance. Methods: One hundred patients with obstructive airway disease on regular CFC aerosol inhaler medication underwent a standardised, structured interview. During the interview patients were asked to use a new HFA aerosol inhaler and three DPIs in random order. Patients’ notions were recorded. Results: Most patients (96) agreed to change from their CFC to the HFA inhaler, of those, only 12 did so with some reservation. Properties (taste, user-friendliness, design) of the HFA inhaler were rated favourably. DPIs represented an acceptable alternative to aerosol inhalers. In fact, 57 patients preferred a DPI over the HFA inhaler. Not all powder devices were equally acceptable. Replacing the CFC inhaler with patients’ preferred alternative devices resulted in a more than 3-fold increase in costs. Conclusion: Concerns about the acceptability of reformulated CFC-free aerosol inhalers are ill founded. However, if given the choice, many patients prefer a DPI over the HFA inhaler. The transition offers an opportunity to review patients’ current treatment and the proficiency of their inhaling technique. Moving to CFC-free inhalers will have revenue implications.

P234 The importance of appropriate reference equations for spirometry: lessons learned from the Cystic Fibrosis Gene Therapy “Run-In” study

Introduction and Objectives The “Run-In” study is an on-going longitudinal, observational study of CF patients assessing outcome measures for a future gene therapy trial. Spirometry is performed at each visit and volumes are converted to % predicted values according to published reference equations; historically these were separate for adults and children. Here, we describe the issues arising from this approach, and highlight the benefit of using a reference source which bridges the transition from child to adulthood. Methods CF subjects (=10 years; FEV1=40% predicted) were recruited from three sites in London and Edinburgh. Visits were undertaken during periods of stability every 3–6 months; data presented here are from the first four visits. Spirometry was performed on an Easyone spirometer. Volumes were converted to % predicted values according to Rosenthal (<18 years) and Quanjer (=18 years) reference equations. The FEV1 raw data were subsequently re-analysed using Stanojevic reference equations, which span all age ranges. Comparisons were made using paired t-tests. Results 191 patients attended visit 1 (mean age 22.7 years, 55% male; 91 patients <18 years). Rosenthal and Quanjer FEV1% predicted values were significantly higher than the Stanojevic values: mean differences 2.8 (95% CI 1.9 to 3.7) for children with Rosenthal equations (p<0.0001), and 2.4 (95% CI 2.1 to 2.8) for adults using Quanjer equations (p<0.0001). 10 patients transitioned between paediatric and adult reference ranges during the study period; the slope of change in their FEV1% over visits 1–4 was significantly greater with Rosenthal/Quanjer references than with Stanojevic (p=0.001) largely due to an artefactual drop when switching from Rosenthal to Quanjer values. As an example, a female patient aged 17.8 years at visit 1 had a drop in absolute FEV1% predicted between visits 1 and 2 of 11% when Rosenthal/Quanjer were used but only 3% with Stanojevic reference values. Conclusions Our results highlight issues raised when separate adult and paediatric spirometry reference ranges are used in longitudinal study. The UK CF Gene Therapy Consortium has adopted the Stanojevic reference source for all spirometry analysis in its ongoing Clinical Programme.

P107 Pulmonary imaging techniques to identify suitable patients and act as outcome measures in the UK CF Gene Therapy Consortium clinical programme

We are conducting a large, longitudinal study to assess outcome measures and identify optimal patients for a multidose trial of CF gene therapy. Two imaging modalities are being employed: radioisotope deposition scans and high resolution CT. Subjects have undergone these scans on a single occasion, whilst clinically stable. The purpose was: Deposition scan—to determine which patients would be most optimal for topical drug delivery and CT—to assess the suitability of various parameters as efficacy measures. Following inhalation of 99mTc-labelled human serum albumin, planar gamma camera images and SPECT were used to assess 3-D deposition. Images were scored both digitally and visually (I- no defects; II- patchy deposition; III- patchy deposition with large defects; IV- grossly abnormal). HRCT scans were scored by two radiologists on a lobar basis for the following: bronchiectasis (extent/severity), airway wall thickening, mucus plugging and gas trapping. 147 deposition scan were available; digital indices (DI) ranged from 34 (best) to 150 (severely abnormal). Visual scores correlated well with DI (R2 0.63; p<0.001) and both were significantly negatively correlated with FEV1% (p<0.01). Nine Grade IV subjects had a mean (SD) FEV1 of 43.9(4.3)%, significantly lower than groups I-III (p<0.01). On the basis of very poor deposition, this group is considered unsuitable to progress to the trial. Others have deposition scans which suggest that the gene therapy product could be delivered at least moderately well; they will be filtered through other inclusion/exclusion criteria. Potentially reversible components of the HRCT scores are being considered as efficacy outcome measures. As an example, power calculations suggest that our anticipated group size (n=100) would have 80% power to detect a change in wall thickness half that seen with intravenous antibiotics in a previous study. In conclusion, lung imaging techniques have both aided us in the identification of patients to take through into our multi-dose trial and are currently under consideration as efficacy outcomes.