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Cystic fibrosis outcome parameters

Abstract:

Introduction Cystic Fibrosis Cystic Fibrosis (CF) is the most common severe hereditary disease in Caucasian populations, affecting approximately 1 in 3500 births. Mutations in the CFTR gene cause abnormal production and/or function of the CFTR protein, an anion channel in the apical membrane with a high expression in most cells lining the airways and the ducts of exocrine glands as the sweat gland, the pancreas or the vas deferens. The abnormal CFTR function results in defective ion and water transport, leading to abnormal viscous respiratory secretions and eventually progressive respiratory insufficiency, high chloride levels in sweat, pancreatic insufficiency, and male infertility. Even with optimal therapy, CF is a life shortening disease with a predicted median survival of around 50 years. Although CF is a multisystem disease, more than 90% of patients die from lung disease.(1) Complex genetics necessitate a personalized medicine approach with genotype-specific disease modifying therapies More than 2000 different mutations in the CFTR gene have been reported in the CFTR1 database.(2) The most frequent mutation is F508del, for which around half of the patients are homozygous. The other patients carry at least one of the numerous other mutations, which are almost all very rare. In each country, only 5 or 6 mutations apart from F508del are found in more than 1% of the patients.(3) The current treatment for CF is mainly symptomatic: improving mucociliary clearance with mucolytics and physiotherapy, treating lung infection with antibiotics and lung transplantation in end stage lung disease. Eventually, more than 20 years after the discovery of the CFTR gene, treatments that aim at correcting the basic CFTR defect are emerging. New ‘disease modifying therapies’ were recently developed to target the basic defect, thereby directly improving or restoring the function of the CFTR protein.(4) Two of these are already on the market, and many more are at varying stages of development. Ivacaftor is a ‘potentiator’ and restores the chloride transport in patients with gating mutations, in which CFTR protein is present at the apical membrane but are not activated. Ivacaftor improves sweat chloride, lung function and weight in patients with gating mutations. Lumacaftor is a ‘corrector’, allowing a proportion of the misfolded F508del protein to traffic to the cell membrane. The combination of lumacaftor and ivacaftor resulted in improvement of the sweat chloride value and of the lung function in patients homozygote for the F508del mutation. The diversity of genetic abnormalities in CF results in the need for different compounds targeting each type of mutation, so-called ‘genotype specific personalized medicine’. Clinical trials in patients homozygous for F508del are feasible because sufficient patient numbers are available. Performing clinical trials to prove the effect of a disease modifying therapy becomes much more challenging in patients carrying rare mutations. And even in patients with less rare mutations, reliable and precise outcome measures give more robust evidence and allow more efficient use of patients and financial means. With the advent of these disease-modifying treatments the need for outcome parameters that accurately quantify the improvement in the underlying disease defect are more than ever important. Clinical trials and the need for sensitive and accurate outcome measures Clinical trials assess the risk/benefit profile of a compound. Phase 1 trials establish the safety and the tolerability of the new drug in healthy volunteers and allow dose finding. Phase 2 trials assess compound safety in patients, and evaluate ‘proof of concept’ of the effect of the drug on the disease process. At this stage, biomarkers are often used. Given the low number of participants in such trials and the short observation period, a significant effect on clinical or surrogate endpoints is often not achievable. In larger-scale phase 3 trials, the beneficial effect of the drug should be demonstrated as a substantial improvement in clinical or surrogate endpoints. Sensitive endpoints, applicable in the population studied, responsive to intervention and adapted to the phase of the trial are thus required. Endpoints or outcome measures can be classified in three categories: - Clinical outcome measures measure how patients feel, function or survive. Increased survival is the result of improvement in the treatment of patients with cystic fibrosis. With a median survival of 35 years, survival is an impossible endpoint to use in clinical trials. Other clinical endpoints are the frequency of respiratory exacerbations and quality of life questionnaires such as the respiratory part of the Cystic Fibrosis Questionnaire-Revised (CFQ-R). - Surrogate outcome measures are lab measurements used as substitutes for clinical end-points. To qualify as surrogate outcome, the link between the measurement and eventual clinical disease outcome such as survival must be established. At present, forced expiratory volume in 1 sec (FEV1) is the only FDA approved surrogate endpoint for clinical trials in CF. - Biomarkers are an objective measure of a normal or pathogenic biologic process, or of a pharmacologic response to a therapeutic intervention. In CF, biomarkers that reflect CFTR function and ion transport are the sweat chloride concentration, the nasal potential difference measurement or intestinal current measurement. Other biomarkers reflect early pathogenic processes, e.g. the mucociliary clearance. A combination of endpoints can be used during the evaluation of a new therapy in patients with CF. Given the importance of accurately assessing new therapies, our research has focused on 2 types of outcome measures. First, outcome measures that reflect the basic CF defect and that are thus especially useful in phase 2 clinical trials with disease modifying drugs: nasal potential difference (NPD) and sweat chloride. Next, outcome measures that reliably quantify CF lung disease, the major cause of death in CF. These parameters are especially useful in phase 3 clinical trials, when proof of clinical benefit must be provided. We explored the value of lung clearance index (LCI) and FEF25-75. Study objectives, methods and results 1.Nasal potential difference A nasal potential difference (NPD) measurement quantifies the voltage across the nasal epithelium resulting from the mucosal ion transport. It directly assesses the CFTR function by measuring chloride transport, taking into account the function of ENaC, a sodium transporter, which is inhibited by CFTR. A catheter is placed in the nose, with a sensing electrode to measure the potential and a channel to perfuse solutions locally on the nasal mucosa. A fixed perfusion sequence is used: Ringer solution and Ringer with amiloride assessing sodium transport through ENaC, followed by a zero-chloride solution and subsequent addition of isoproterenol. The change in potential induced by the latter two solutions is called the total chloride response (TCR) and is a measure of chloride transport through CFTR. The NPD differentiates patients with CF from healthy controls. Patients with CF and mild phenotypes show intermediate NPD values reflecting the degree of residual CFTR function.(5) NPD has been used as an exploratory endpoint in trials, measuring the change in CFTR function during treatment with disease modifying therapies. With ivacaftor, a dose response was observed: progressive improvement of chloride transport with increasing drug doses.(6) The NPD is thus a unique way to measure CFTR function in the airways, reflecting the first step of the pathophysiological cascade initiating the respiratory illness responsible for most of the morbidity and mortality of CF. The challenges with NPD as endpoint are the lack of standardization and the poor repeatability of the measurements. Therefore, we investigated the impact of several technical aspects of the NPD measurement, with an emphasis on improving repeatability to reduce the sample size in clinical trials. Vermeulen, F., Proesmans, M., Feyaerts, N., & De Boeck, K. (2011). Nasal potential measurements on the nasal floor and under the inferior turbinate: does it matter? Pediatr Pulmonol, 46(2), 145-152. Nasal potential is not uniform over the nasal mucosa. Therefore, the place of the catheter in the nostril could have an impact on the results obtained. We examined the influence of the place of the catheter by comparing the values obtained with the nasal catheter placed medially on the nasal floor in one nostril (the ‘European’ floor protocol), and laterally under the inferior turbinate in the opposite nostril (the ‘American’ turbinate protocol). Thirty-four patients with CF, 26 heterozygotes and 61 control subjects underwent simultaneous measurements with both techniques, with a repeat test in 57 to measure repeatability. Both protocols discriminated well between CF and control subjects, and the mean NPD values were not different. Repeatability was similar with the two methods. Sample size projections using the proportion of interpretable measurements, and mean values in CF and in control subjects slightly favored the use of the ‘floor catheter’ (Table 1). Bronsveld, I.*,Vermeulen, F.*, Sands, D., Leal, T., Leonard, A., Melotti, P., European Cystic Fibrosis Society - Diagnostic Network Working, G. (2013). Influence of perfusate temperature on nasal potential difference. Eur Respir J, 42(2), 389-393. (*First authors equal contributions) The effect of the temperature of the perfused solutions was evaluated. Different devices were in use to warm the solutions to 34-37°C during local perfusion, while some operators used solutions at room temperature. To assess the effect of the temperature of the solutions, NPD was measured in healthy subjects (CF subjects have virtually absent chloride transport) at different perfusion temperatures: Warmed (34-37°C, W) or room temperature (RT). Two sequences were applied in random order: RT-W-RT and W-RT-W. Temperature changes during perfusion had a minor effect on NPD (from -1.4+/- 3.7 to mV to +2.1 +/- 4.4 mV). Complete NPD tracings obtained with warmed and room temperature solutions in 24 healthy subjects were compared, showing no significant effect of solution temperature on NPD values. We concluded that warming was not essential for NPD measurements, allowing a simplified operating procedure that was included in the last version of the joint SOP of the European and American clinical trial networks. Vermeulen, F., Proesmans, M., Boon, M., & De Boeck, K. (2015). Improved repeatability of nasal potential difference with a larger surface catheter. J Cyst Fibros, 14(3), 317-323. To improve repeatability, we then designed a custom NPD catheter with a larger contact area than the ‘standard’ catheters (Figure 3), thereby improving catheter stability and allowing measurement over a large surface. This results in a ‘mean’ rather than a ‘point’ measurement, which is expected to decrease variability, as the NPD is not uniform throughout the nostril. SOPs prescribe to start the measurement after taping the catheter at the location with the most negative potential. Aiming at a fixed rather than a variable position for the catheter should further decrease test-to-test variability. NPD values obtained with the custom catheter were thus compared to values obtained with the ‘standard’ catheters in 394 tracings. Use of the ‘custom’ large surface catheter resulted in slightly lower total chloride responses, but in a much-improved repeatability. Taking the measurement at a fixed location, rather than from the location with the highest basal potential also improved repeatability. Sample sizes projections using change in total chloride responses and repeatability of the measurement showed that the use of the large surface catheter at a fixed location allows to reduce the number of subjects to include in a trial to around 50%, compared with a thin catheter at the location with the most negative basal potential as prescribed in the SOP. We also joined multicentric evaluations of NPD measurements: an agar-filled catheter performed better than catheters perfused with a conducting solution. A study evaluated a quality control process for interpretation of NPD tracings in clinical trials. 2.Sweat Chloride After stimulation of sweat secretion by pilocarpine iontophoresis, the chloride concentration in sweat is measured. The sweat test is the gold standard for diagnosis of CF, with clear cutoffs to differentiate CF subjects and controls.(7) When sweat chloride is below 30 mmol/L CF is unlikely, above 60 mmol/L is diagnostic for CF, and between 30 and 60 mmol/L is indeterminate and requires further diagnostic testing (genetic analysis and physiologic tests of CFTR function). The well standardized and non-invasive collection procedure makes sweat chloride very appealing as endpoint, and it has been used extensively as a biomarker of CFTR function in clinical trials of disease modifying therapies.(8) CFTR modulators improved sweat chloride, to near-normal values with the highly efficient ivacaftor in patients with gating mutations.(6) However, little is known about the natural variability of sweat chloride over time in patients with CF. Knowledge of this variability is crucial for sample size calculations and design of clinical trials. We therefore assessed the variability of sweat chloride in two settings: diagnostic sweat tests and a clinical trial. Vermeulen, F., Lebecque, P., De Boeck, K., & Leal, T. (2017). Biological variability of the sweat chloride in diagnostic sweat tests: A retrospective analysis. J Cyst Fibros, 16(1), 30-35. We assessed the variability of sweat chloride in a retrospective analysis of 5904 sweat tests performed in two CF reference centers over a 14 year period. Within test limits of repeatability (difference between sweat chloride in samples from right and left arm on the same test occasion) were between -3.2 and +3.6 mmol/L in 1022 tests, with similar diagnostic conclusion from both arms in all but 3 tests. Between tests limits of repeatability (difference between results from two tests occasions) were larger, between ‑18 and +14 mmol/L in the 197 subjects with two valid tests. The diagnostic conclusions remained the same for almost all tests in the CF or in the normal range. For half of the subjects with a result in the intermediate range, sweat chloride was normal on repeat testing. When the second test was also in the intermediate range, CFTR mutations were found more often. Vermeulen, F., Le Camus, C., Davies, J. C., Bilton, D., Milenkovic, D., & De Boeck, K. (2017). Variability of sweat chloride concentration in subjects with cystic fibrosis and G551D mutations. J Cyst Fibros, 16(1), 36-40. We analyzed the data from the placebo arm of a phase 3 trial to measure variability of sweat chloride in a clinical trial setting. In 78 patients with CF and a G551D mutation, sweat chloride was measured on 8 test occasions over 48 weeks. The variance component analysis showed that variance was mainly between subjects (SD 8.9 mmol/L) and within subject (SD 8.1 mmol/L), with a lower within test variance (SD 4.8 mmol/L). Using a crossover design to reduce between-subject variability, the average of repeated measurements to decrease within-patient variability and the average of the collections of both arms to decrease within-test variability allows to lower the sample size for clinical trials of CFTR modulators (Table 3). With with a drug anticipated to improve sweat chloride by at least 15 mmol/L, averaging 4 bilateral measurements in each of the periods of a crossover trial allows reach a power of 90% in a trial including 5 patients. 3.Lung Clearance Index and FEF25-75 The only validated surrogate outcome in CF trial is the FEV1, measured by spirometry. FEV1 correlates with survival. However, FEV1 has become an insensitive outcome, especially in children and in adult patients with mild disease: it remains normal until far into adolescence and decline rates have become very small. Early lung disease in CF is thought to affect the small airways, also called ‘the silent zone’ because conventional respiratory function tests measuring flows or resistance in the airways are insensitive to detect abnormalities in this zone. The lung clearance index does not rely on measurement of flow and resistance but on quantification of the inhomogeneity of the ventilation arising from small airways disease. A tracer gas (such as helium, nitrogen or SF6) is first washed in until a stable concentration is reached in the lungs. Then the tracer gas is washed out of the lungs, breath after breath. The total expired volume needed to lower the tracer gas concentration to 1/40th of the starting value, corrected for the functional residual capacity (the lung volume at rest), is the LCI. A high LCI reflects poor gas mixing in the lungs resulting mainly from abnormalities in the small airways, the site of early lung damage in CF.(9) Unlike FEV1, LCI is almost independent from age, gender and body size in school-age children and young adults, making it an ideal parameter for longitudinal follow up.10 In patients with CF, the measurement of LCI is feasible and repeatable and is more sensitive than FEV1 to detect early CF lung disease. An abnormal LCI correlates with more structural lung damage assessed by CT imaging. An abnormal LCI in toddlers predicts abnormal spirometry at school age.10 LCI is also responsive to intervention, as it improves after treatment with hypertonic saline, RhDNAse or CFTR modulators.11 Before promotion as a surrogate outcome measure, more information is needed about long term changes of LCI and its correlation with clinical endpoints. FEF25-75, the flow between 25 and 75% of the vital capacity of the lungs is a measure of small airways disease based on forced expiration. There is some debate whether FEF25-75 could also be more sensitive to early lung disease in patients with CF. Vermeulen, F., Ophoff, J., Proesmans, M., & De Boeck, K. (2013). Comparison of lung clearance index measured during helium washin and washout in children with cystic fibrosis. Pediatr Pulmonol, 48(10), 962-969. We measured LCI in the children followed at the CF center of Leuven. Most of the early publications on LCI in CF report results obtained with a research setup measuring the washout of the tracer gas SF6 with a mass spectrometer. This complex setup is impossible to use in multicentric trials, and therefore we performed the MBW with an early commercially available device based on ultrasonic molar mass measurements and using helium as tracer gas. With this device, we established LCI values in a cohort of 65 healthy children and 65 children with CF. We assessed whether the LCI measured during the washout (WO) of He was correlated with a comparable measurement during the washin (WI), as this would allow to shorten the measurement time by performing only a washin rather than a washin and a washout. Results calculated from the WI and from the WO were only weakly correlated (R=0.440, p<0.001) in patients with CF. This study highlighted some limitations of the helium measurement setup, which led us to switch to a commercial nitrogen washout setup, that would later become generalized in the CTN-centers. Vermeulen, F., Proesmans, M., Boon, M., Havermans, T., & De Boeck, K. (2014). Lung clearance index predicts pulmonary exacerbations in young patients with cystic fibrosis. Thorax, 69(1), 39-45. We subsequently started a prospective, longitudinal observational study to follow LCI on the long term in children with CF and to correlate LCI with other endpoints, such as pulmonary exacerbations (PE), chest CT scan, spirometry and a validated patient-reported outcome (PRO), the Cystic Fibrosis Questionnaire-Revised (CFQ-R). In a first study, the predictive value of baseline LCI for a PE in the next year was demonstrated. In 63 patients with CF aged 5-19 years, a higher baseline LCI was associated with a shorter time to pulmonary exacerbation and a higher rate of exacerbation. An increase in the LCI z-score of 1 resulted in an increase in the PE rate by 12.0% (95% CI 5.0% to 19.5%, p=0.001). LCI and CFQ-Rresp were correlated. In the subgroup of 53 patients with a normal baseline FEV1, LCI still predicted pulmonary exacerbations (Figure 5) and correlated with CFQ-Rresp, while FEV1 did not, showing the superiority of LCI as early disease marker. FEF25-75 was also assessed as potentially more sensitive to changes in the small airways and thus to early disease in CF. FEF25-75 and FEV1 were strongly correlated (Rsp 0.731, p<0.001). Of the 53 patients with normal FEV1, 47 (89%) also had a normal FEF25-75, while LCI was abnormal in 42, pointing out that FEF25-75 adds little information compared to FEV1 to detect early lung disease. FEF25-75 did not perform better than FEV1 to predict pulmonary exacerbations, nor did it correlate better with CFQ-Rresp. In the second part of this study, we measured the changes in LCI over longer periods and assessed predictors of a more rapid worsening of the LCI. The first interim analysis included 36 children with CF, who performed a median of 6 LCI measurements over a mean follow-up of 3.1 year and a chest CT close to the first and the last LCI measurement. Certified readers quantified structural damage on the chest CT scan using the CFCT score. LCI worsened in 28 of the 36 patients, with a median increase of +0.5/year. The increase was higher in the youngest patients (+0.8/year before 8 years vs +0.4/year after 8 years, p=0.019). LCI increased less in patients with a normal CFCT score (+0.1/year) than with an abnormal CFCT score (+0.7/year, p=0.05). LCI at baseline, FEV1 at baseline and treatment with IV antibiotics during follow-up were not associated with LCI changes. Changes in LCI and in in FEV1 were correlated (Rsp=-0.608, p<0.001), as were changes in LCI and in CFCT scores (Rsp=0.537, p=0.001). A correlation was also found between changes in CFCT scores and in FEV1. The cohort will be further extended when more CFCT scans are available for scoring. Conclusion We explored important aspects of outcome parameters needed to assess the effect of innovative disease-modifying therapies for cystic fibrosis. As most therapies are mutation specific, a personalized medicine approach will be necessary in the many patients with rare mutations. Outcome parameters sensitive to interventions and with low variability will be required to assess the effect of the drugs in these small groups of patients. We refined technical aspects of the nasal potential difference measurements to come to an optimized measurement protocol, reducing the variability of the nasal potential difference and making it more suitable for clinical trials with low number of patients. After defined the variability of sweat chloride, we proposed an optimal trial design with repeated measurements, as a contribution to optimal planning of clinical trials. Lung clearance index is currently the most promising parameter to measure early lung function abnormalities. In patients with normal spirometry, it will likely be the only feasible way to monitor lung disease on the long term. We established an important link between LCI as biomarker of lung disease and clinical endpoints such as pulmonary exacerbations and a patient-reported outcome. This will contribute to the promotion of LCI to surrogate endpoint. Quantification of the long term LCI changes in patients and of the link between LCI changes and changes in other parameters will contribute to better planning of future studies using LCI as endpoint. References 1. Stoltz, D.A., Meyerholz, D.K. & Welsh, M.J. Origins of cystic fibrosis lung disease. N Engl J Med 372, 1574-5 (2015). 2. Cystic Fibrosis Genetic Analysis Consortium, http://www.genet.sickkids.on.ca/app. 3. De Boeck, K., Zolin, A., Cuppens, H., Olesen, H.V. & Viviani, L. The relative frequency of CFTR mutation classes in European patients with cystic fibrosis. J Cyst Fibros 13, 403-9 (2014). 4. Bell, S.C., De Boeck, K. & Amaral, M.D. New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls. Pharmacol Ther 145, 19-34 (2015). 5. Wilschanski, M. et al. Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials. Am J Respir Crit Care Med 174, 787-94 (2006). 6. Accurso, F.J. et al. Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. N Engl J Med 363, 1991-2003 (2010). 7. Farrell, P.M. et al. Diagnosis of Cystic Fibrosis: Consensus Guidelines from the Cystic Fibrosis Foundation. J Pediatr 181S, S4-S15 e1 (2017). 8. Muhlebach, M.S. et al. Biomarkers for cystic fibrosis drug development. J Cyst Fibros 15, 714-723 (2016). 9. Horsley, A.R. et al. Lung clearance index is a sensitive, repeatable and practical measure of airways disease in adults with cystic fibrosis. Thorax 63, 135-40 (2008). 10. Robinson, P.D. et al. Consensus statement for inert gas washout measurement using multiple- and single- breath tests. Eur Respir J 41, 507-22 (2013). 11. Davies, J. et al. Assessment of clinical response to ivacaftor with lung clearance index in cystic fibrosis patients with a G551D-CFTR mutation and preserved spirometry: a randomised controlled trial. Lancet Respir Med 1, 630-8 (2013).