Julie Avolio

Inconclusive Diagnosis of Cystic Fibrosis After Newborn Screening

OBJECTIVES: To prospectively study infants with an inconclusive diagnosis of cystic fibrosis (CF) identified by newborn screening (NBS; “CF screen positive, inconclusive diagnosis” [CFSPID]) for disease manifestations. METHODS: Infants with CFSPID and CF based on NBS from 8 CF centers were prospectively evaluated and monitored. Genotype, phenotype, repeat sweat test, serum trypsinogen, and microbiology data were compared between subjects with CF and CFSPID and between subjects with CFSPID who did (CFSPID→CF) and did not (CFSPID→CFSPID) fulfill the criteria for CF during the first 3 years of life. RESULTS: Eighty-two subjects with CFSPID and 80 subjects with CF were enrolled. The ratio of CFSPID to CF ranged from 1:1.4 to 1:2.9 in different centers. CFTR mutation rates did not differ between groups; 96% of subjects with CFSPID and 93% of subjects with CF had 2 mutations. Subjects with CFSPID had significantly lower NBS immunoreactive trypsinogen (median [interquartile range]:77 [61–106] vs 144 [105–199] μg/L; P < .0001) than did subjects with CF. Pseudomonas aeruginosa and Stenotrophomonas maltophilia were isolated in 12% and 5%, respectively, of subjects with CFSPID. CF was diagnosed in 9 of 82 (11%) subjects with CFSPID (genotype and abnormal sweat chloride = 3; genotype alone = 4; abnormal sweat chloride only = 2). Sweat chloride was abnormal in CFSPID→CF patients at a mean (SD) age of 21.3 (13.8) months. CFSPID→CF patients had significantly higher serial sweat chloride (P < .0001) and serum trypsinogen (P = .009) levels than did CFSPID→CFSPID patients. CONCLUSIONS: A proportion of infants with CFSPID will be diagnosed with CF within the first 3 years. These findings underscore the need for clinical monitoring, repeat sweat testing at age 2 to 3 years, and extensive genotyping.

β-Adrenergic Sweat Secretion as a Diagnostic Test for Cystic Fibrosis

RATIONALE β-Adrenergically induced sweat secretion offers an expedient method to assess native cystic fibrosis transmembrane conductance regulator (CFTR) secretory function in vivo. OBJECTIVES To evaluate the sensitivity, specificity, and reliability of a test based on the activity and secretory function of CFTR in the sweat gland. METHODS Primary and validation trials with prospectively ascertained healthy control subjects, obligate heterozygotes, and patients with a CFTR-related disorder and CF (pancreatic sufficient and insufficient). MEASUREMENTS AND MAIN RESULTS Diagnostic accuracy and reliability of β-adrenergic sweat secretory rates using an evaporimeter was assessed and compared with sweat chloride concentrations. The cholinergically stimulated mean sweat rate did not differ among groups. The mean maximal β-adrenergically stimulated sweat rate in heterozygotes was about half the rate of healthy control subjects, and completely absent in pancreatic-insufficient patients with CF and pancreatic-sufficient patients with CF (P < 0.0001). Subjects with a CFTR-related disorder showed reduced or absent β-adrenergic sweat secretion. The β-adrenergic secretory response demonstrated high diagnostic accuracy (area under a characteristic receiver-operator curve = 0.99; 95% confidence interval, 0.97-1.00) and reliability (intraclass correlation, 0.90; 95% confidence interval, 0.81-0.95). The diagnostic cutoff level for CF, derived from the primary trial, correctly identified all control subjects, heterozygotes, and patients with CF in the validation cohort, whereas concurrent sweat chloride measurements misclassified one heterozygote and five subjects with CF. The cholinergic and β-adrenergic sweat secretion rates were lower in women compared with men (P < 0.001). CONCLUSIONS β-Adrenergic sweat secretion rate determined by evaporimetry is an accurate and reliable technique to assess different levels of CFTR function and to identify patients with CF.

Effect of ivacaftor therapy on exhaled nitric oxide in patients with cystic fibrosis

UNLABELLED Airways of patients with cystic fibrosis are deficient for nitric oxide. Low nitric oxide in cystic fibrosis has been shown to be associated with poor pulmonary function and risk of infection with certain pathogens. Treatment of cystic fibrosis patients with the cystic fibrosis transmembrane conductance regulator (CFTR)-targeting drug ivacaftor results in improved pulmonary function. The effect of ivacaftor on airway nitric oxide has not been assessed. METHODS In this observational trial, fractional exhaled nitric oxide (FE(NO)) was measured before and 4 weeks after initiation of ivacaftor therapy, in patients with cystic fibrosis and a CFTR gating mutation. The effect of ivacaftor on FE(NO) was compared to treatment with inhaled dornase alfa or hypertonic saline for 4 weeks, respectively. RESULTS A total of 15 patients on ivacaftor therapy were studied. Pulmonary function improved significantly and mean (±SD) FE(NO) increased from 8.5±5.0 to 16.2±15.5 ppb. The effect was more pronounced in pediatric compared to adult patients. There was no linear correlation between changes in FE(NO), pulmonary function or sweat chloride concentration. Neither treatment with inhaled dornase alfa (n=15) or hypertonic saline (n=16) resulted in a change in FE(NO). CONCLUSION Therapy with ivacaftor results in an increase in nitric oxide formation in cystic fibrosis airways, while dornase alfa or hypertonic saline has no effect on airway nitric oxide. Some beneficial effects of CFTR targeting therapy in CF may result from improved airway nitric oxide production.

Phenotypic profiling of CFTR modulators in patient-derived respiratory epithelia

Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype–phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the “gold-standard” but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures.Cystic fibrosis: toward personalized therapiesA new method for evaluating drug responses in patient-derived respiratory tissue promises to help determine the best treatment for each patient with cystic fibrosis (CF). CF patients are highly susceptible to lung infections due to the build-up of thick mucus in the airways. Over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified in patients with CF, which partly explains their varied response to treatment. Saumel Ahmadi, Christine E. Bear, and colleagues at the Hospital for Sick Children in Toronto developed a fluorescence-based method for measuring improvements in mutant CFTR function in patient-derived nasal and induced pluripotent stem cell-derived lung tissue. This method enables comparison of approved and investigational drugs on airway cells from each individual patient and in the longer term will accelerate the development of personalized therapeutic strategies.

Lung clearance index response in patients with CF with class III CFTR mutations

Ivacaftor (KALYDECO) is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that increases transmembrane chloride flux in vitro and leads to significant benefits in patients with cystic fibrosis (CF) with class III gating mutations.1–5 Ivacaftor is associated with sustained improvement in FEV1 and weight as well as reduced time to next pulmonary exacerbation.5–7 It has also been shown that 4 weeks of ivacaftor improves the lung clearance index (LCI) in patients with CF with preserved lung function.8 It is presently unclear whether LCI, a measure of ventilation inhomogeneity, provides additional information among patients with more impaired lung function as well as whether the sustained effectiveness of ivacaftor as demonstrated by improvements in the previously mentioned outcomes is also evident in the LCI response. The aim of this observational study was to assess the LCI before and after initiation of ivacaftor treatment over 6 months in patients with CF with a wider range of …

Nasal potential difference: Best or average result for CFTR function as diagnostic criteria for cystic fibrosis?

BACKGROUND The current practice of averaging the nasal potential difference (NPD) results of right and left nostril measurements reduce inter-individual variability but may underestimate individual CFTR function. METHODS Best NPD response to Cl(-)-free and isoproterenol perfusion (=largest ΔPD(0Cl/Iso)) from the right and left nostril was compared to the average result in 13 cystic fibrosis (CF), 78 query-CF patients and 22 healthy controls from 2 cohorts. RESULTS Despite moderate to good correlation (p<0.001) between right and left measured ΔPD(0Cl/Iso), we observed large differences in some individuals. A comparison of average versus best ΔPD(0Cl/Iso) showed only moderate agreement (Giessen κ=0.538; Toronto κ=0.607). Averaging ΔPD(0Cl/Iso) showed a lower composite chloride response compared to best ΔPD(0Cl/Iso) and altered diagnostic NPD interpretation in 30 of 113 (27%) subjects. CONCLUSIONS The current practice of averaging the NPD results of right and left nostril measurements leads to an underestimation of the individual CFTR function and should be reconsidered.

Testing gene therapy vectors in human primary nasal epithelial cultures

Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF.

WS14.6 Beta-adrenergic sweat secretion rate as accurate biomarker for CFTR function

WS14.5 A functional CFTR assay using primary cystic fibrosis intestinal organoids J.F. Dekkers1,2,3, C.L. Wiegerinck2,4, H.R. de Jonge5, N.W.M. de Jong1,2,3, M.J.C. Bijvelds5, E.E. S Nieuwenhuis4, S. van den Brink6, H. Clevers6, C.K. van der Ent1, S. Middendorp2,4, J.M. Beekman1,2,3. 1Dept of Pediatric Pulmonology, 2Center for Molecular and Cellular Intervention, 3Dept of Immunology, 4Dept of Pediatric Gastroenterology, Wilhelmina Children’s Hospital, University Medical Centre, Utrecht, The Netherlands; 5Dept of Gastroenterology & Hepatology, Erasmus MC-University Medical Center, Rotterdam, The Netherlands; 6Hubrecht Institute for Developmental Biology and Stem Cell Research, and University Medical Center Utrecht, The Netherlands

144 Effect of treatment with ivacaftor on exhaled nitric oxide

Objectives Nitric oxide (NO) is reduced in cystic fibrosis (CF) airways and NO deficiency may contribute to CF lung disease. Previous studies have shown that antibiotic treatment for CF pulmonary exacerbations resulted in an increase in airway NO formation. Ivacaftor is the first CFTR-targeting drug that was approved for the treatment of people with CF and certain CFTR gating mutations. Methods We studied the effect of ivacaftor treatment on airway NO in people with CF in an observational trial. The study was approved by the pediatric and adult study site Institutional Research Ethics Boards. Results So far, a total of 12 (8 pediatric and 4 adult) patients with CF were recruited. All carried at least one copy of the Gly551Asp-CFTR mutation. Mean age at enrolment was 20 (range 6–50) years. Pulmonary function (spirometry) and fraction of exhaled NO (FENO 50 ) were measured before and 4 weeks after initiation of ivacaftor treatment. Both mean (±SD) forced vital capacity (93.4±11.6 vs 100.9±9.1% of predicted, p=0.004) and forced expiratory volume in one second (FEV1) (71.6±16.0 vs 83.3±16.2% of predicted, p=0.002) improved with treatment. FENO increased in all but one of the study participants (p Conclusion Four weeks of treatment with ivacaftor resulted in a significant increase in pulmonary function and airway NO formation. These data suggest that CFTR-targeting therapies may result in reconstitution of impaired airway NO formation in patients with CF.