Detection of cystic fibrosis transmembrane conductance regulator activity in early-phase clinical trials

Abstract

Advances in our understanding of cystic fibrosis pathogenesis have led to strategies directed toward treatment of underlying causes of the disease rather than treatments of disease-related symptoms. To expedite evaluation of these emerging therapies, early-phase clinical trials require extension of in vivo cystic fibrosis transmembrane conductance regulator (CFTR)-detecting assays to multicenter trial formats, including nasal potential difference and sweat chloride measurements. Both of these techniques can be used to fulfill diagnostic criteria for the disease, and can discriminate various levels of CFTR function. Full realization of these assays in multicenter clinical trials requires identification of sources of nonbiological intra- and intersite variability, and careful attention to study design and statistical analysis of study-generated data. In this review, we discuss several issues important to the performance of these assays, including efforts to identify and address aspects that can contribute to inconsistent and/or potentially erroneous results. Adjunctive means of detecting CFTR including mRNA expression, immunocytochemical localization, and other methods are also discussed. Recommendations are presented to advance our understanding of these biomarkers and to improve their capacity to predict cystic fibrosis outcomes.

Figure 1.

Representative nasal potential difference (PD) tracing and scoring methods. (A) Representative basal PD measurement of a normal subject. Basal PD can be interpreted as the most polarized (maximal) basal measure (e.g., 29 mV at 2 cm) or the mean of the five individual measures (0.5, 1.0, 1.5, 2.0, and 3.0 cm are designated). AT represents measurement of PD at the anterior tip of the inferior turbinate (a standardized measure to ensure reproducibility). (B) Perfusion tracing from a normal subject. The x axis indicates time (min) and the y axis represents the potential difference (mV, polarizing). Initiation of various perfusion solutions is designated by arrows (bottom). Sodium transport can be quantified by Ringer's perfusion (about 20 mV) or by a change after amiloride (100 μM) perfusion (ΔAmil). Chloride transport is quantified by the change in PD after zero chloride perfusion (Δ0 Cl⁻), the change after isoproterenol (10 μM) or other cystic fibrosis transmembrane conductance regulator (CFTR) agonist (ΔIso), or the sum of these changes (total chloride secretion [TCS], shown in red). Both sodium and chloride transport can be captured in a single measure by the total change in PD (ΔPD, shown in red). (C) Perfusion tracing from a subject with CF. Compared with the tracing from a normal subject, the CF tracing has a relatively polarized PD in Ringer's, a large depolarization with amiloride, and no sustained repolarization with zero chloride and isoproterenol solutions. The large depolarization after ATP perfusion serves as a positive control. Measures for quantifying sodium and chloride transport are also shown.

Figure 1.

Representative nasal potential difference (PD) tracing and scoring methods. (A) Representative basal PD measurement of a normal subject. Basal PD can be interpreted as the most polarized (maximal) basal measure (e.g., 29 mV at 2 cm) or the mean of the five individual measures (0.5, 1.0, 1.5, 2.0, and 3.0 cm are designated). AT represents measurement of PD at the anterior tip of the inferior turbinate (a standardized measure to ensure reproducibility). (B) Perfusion tracing from a normal subject. The x axis indicates time (min) and the y axis represents the potential difference (mV, polarizing). Initiation of various perfusion solutions is designated by arrows (bottom). Sodium transport can be quantified by Ringer's perfusion (about 20 mV) or by a change after amiloride (100 μM) perfusion (ΔAmil). Chloride transport is quantified by the change in PD after zero chloride perfusion (Δ0 Cl⁻), the change after isoproterenol (10 μM) or other cystic fibrosis transmembrane conductance regulator (CFTR) agonist (ΔIso), or the sum of these changes (total chloride secretion [TCS], shown in red). Both sodium and chloride transport can be captured in a single measure by the total change in PD (ΔPD, shown in red). (C) Perfusion tracing from a subject with CF. Compared with the tracing from a normal subject, the CF tracing has a relatively polarized PD in Ringer's, a large depolarization with amiloride, and no sustained repolarization with zero chloride and isoproterenol solutions. The large depolarization after ATP perfusion serves as a positive control. Measures for quantifying sodium and chloride transport are also shown.

Figure 1.

Representative nasal potential difference (PD) tracing and scoring methods. (A) Representative basal PD measurement of a normal subject. Basal PD can be interpreted as the most polarized (maximal) basal measure (e.g., 29 mV at 2 cm) or the mean of the five individual measures (0.5, 1.0, 1.5, 2.0, and 3.0 cm are designated). AT represents measurement of PD at the anterior tip of the inferior turbinate (a standardized measure to ensure reproducibility). (B) Perfusion tracing from a normal subject. The x axis indicates time (min) and the y axis represents the potential difference (mV, polarizing). Initiation of various perfusion solutions is designated by arrows (bottom). Sodium transport can be quantified by Ringer's perfusion (about 20 mV) or by a change after amiloride (100 μM) perfusion (ΔAmil). Chloride transport is quantified by the change in PD after zero chloride perfusion (Δ0 Cl⁻), the change after isoproterenol (10 μM) or other cystic fibrosis transmembrane conductance regulator (CFTR) agonist (ΔIso), or the sum of these changes (total chloride secretion [TCS], shown in red). Both sodium and chloride transport can be captured in a single measure by the total change in PD (ΔPD, shown in red). (C) Perfusion tracing from a subject with CF. Compared with the tracing from a normal subject, the CF tracing has a relatively polarized PD in Ringer's, a large depolarization with amiloride, and no sustained repolarization with zero chloride and isoproterenol solutions. The large depolarization after ATP perfusion serves as a positive control. Measures for quantifying sodium and chloride transport are also shown.

Figure 2.

Effect of variance on number of subjects required for NPD studies. Data from a Cystic Fibrosis Foundation Therapeutics Development Network multicenter study (as described in Table 1) were used to generate the curves shown in Figure 2. The total numbers of study subjects required in each treatment arm to demonstrate a between-group effect (A) (e.g., placebo vs. active therapy) and a within-group effect (B) (e.g., crossover trial) are plotted versus the difference in total CFTR-dependent chloride secretion (polarizing total chloride secretion [TCS], in mV). Each curve represents the number of subjects required as predicted by treatment effect variance (as opposed to the SD of all individual TCS measures summarized in Table 1). The open boxes represent data derived assuming a change in TCS SD of 3.7, as seen in sites using the SOP (n = 5); solid boxes represent data derived assuming an SD of 4.2, as measured when all sites (n = 6) were included. For these calculations, α was 0.05 and power was 0.80.

Figure 3.

Sweat chloride levels versus predicted CFTR activity. Data are extracted from References 10, 79, and 82. CFTR activity is predicted by genotype–phenotype relationships and in vitro studies. Normal individuals are assumed to have 100% CFTR activity. Carriers are assumed to have 50% CFTR activity. Sweat chloride levels are more elevated in patients with two mutations that are usually associated with pancreatic insufficiency (PI) compared with patients who carry two mutations, one of which has been associated with pancreatic sufficiency (PS). Patients with two mutations and congenital absence of the vas deferens (CBAVD) as their sole clinical manifestation of CFTR mutations have sweat chloride levels intermediate between those of patients with PS and carriers. Data are expressed as mean values ± 1 SD.

Figure 4.

The Macroduct sweat collection system. After stimulation of sweat glands by pilocarpine iontophoresis, sweat is collected in a coil. Blue dye indicates the leading edge of sweat collection, providing an estimate of volume of sweat collected during the procedure. Sweat is obtained directly for analysis without the need for elution.