The BATTLE study: Effects of long-term tobramycin inhalation solution (TIS) once daily on exacerbation rate in patients with non-cystic fibrosis bronchiectasis. Study protocol of a double blind, randomized, placebo-controlled trial: study protocol.

Background: Patients with bronchiectasis typically suffer from chronic symptoms such as a productive cough with or without exacerbations leading to hospitalization, causing reduced quality of life (QoL) and mortality. Long-term inhaled antibiotics to treat chronic bronchial infection is registered for use in cystic fibrosis (CF) bronchiectasis. However, in patients with non-CF bronchiectasis data on long-term antibiotics are limited. Objective: To investigate the effectiveness of maintenance tobramycin inhalation solution (TIS) in bronchiectasis patients without cystic fibrosis. Study design: The BATTLE study is a randomized, double blind placebo controlled, multicenter study in the Netherlands performed in patients aged ≥18-year-old with confirmed bronchiectasis, at least two exacerbations in the preceding year, and minimal one positive sputum culture with gram negative pathogens or Staphylococcus aureus, sensitive to tobramycin in the preceding year and at baseline. Patients will be treated with TIS once daily (OD) or placebo (saline 0.9%) OD for 52 weeks followed by a run-out period of 4 weeks after the last dose. The primary outcome is the yearly rate of pulmonary exacerbations. Among secondary outcome parameters are time to exacerbation, lung function, QoL, microbiological evaluation and safety. Discussion: The BATTLE study is designed to determine the efficacy and safety of maintenance TIS OD in bronchiectasis patients colonized by different pathogens and could lead to important new evidence for TIS therapy in this population.The BATTLE study is registered in Clinical trials.gov with registration number: NCT02657473.

ECFS standards of care on CFTR-related disorders: Diagnostic criteria of CFTR dysfunction.

The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD : (1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant.

Females with cystic fibrosis have a larger decrease in sweat chloride in response to lumacaftor/ivacaftor compared to males.

AIM: To explore which patient-related factors influence sweat test response to CFTR modulators, as well as examining the correlation between the sweat chloride response and ppFEV1 or BMI response, using systematically collected real-life clinical data. METHODS: 160 CF patients were identified who had used lumacaftor/ivacaftor for at least six months. Of these patients, age, sweat chloride levels, ppFEV1 weight and BMI at the start of treatment and after 6 months were collected retrospectively. Pearson and Spearman tests were performed to assess correlations. RESULTS: Females compared to males in this group showed a larger response in sweat chloride (mean difference 10.6 mmol/l, 95% CI: 5.7-15.4) and BMI (mean difference 0.27 kg/m2, 95% CI: 0.01-0.54). A modest but significant correlation was found between patient weight and sweat chloride response (Pearson R = 0.244, p = 0.001), which diminished upon correction for the other factors. The correlation between sex and sweat chloride response remained; R = 0.253, p = 0.001. Sweat chloride response did not correlate with ppFEV1 change or BMI change at 6 months after start of therapy. CONCLUSION: Sweat chloride response is larger in females compared to males, which also explains the negative correlation of weight with the response in sweat chloride concentration after start of lumacaftor/ivacaftor. Sweat chloride response does not correlate with the responses in ppFEV1 and BMI. This information may help the interpretation of sweat test results acquired for the follow up and evaluation of CFTR modulating treatments, and warrants further investigation into the underlying mechanisms of sex differences in response to CFTR modulators.

Relationship between lung function and Modified Shuttle Test performance in adult patients with cystic fibrosis: a cross-sectional, retrospective study.

OBJECTIVES: To investigate the relationship between lung function and exercise capacity in adults with cystic fibrosis (CF), and to develop a CF-specific equation to predict Modified Shuttle Test (MST) performance from baseline data. DESIGN: Cross-sectional, retrospective study. SETTING: Adult CF centre. PARTICIPANTS: One hundred and twenty-seven patients with CF [61 male; mean age 25 years (range 17 to 52 years), mean forced expiratory volume in 1second (FEV1) 56% predicted (range 15 to 124%)]. MAIN OUTCOME MEASURES: MST and FEV1. RESULTS: Overall, a moderate-to-good relationship was found between lung function and MST performance (walking distance vs FEV1% predicted: r=0.64, P=0.01). This relationship between FEV1 and MST shows an obvious threshold at an FEV1 of 67% predicted. Above this threshold, no significant association was observed between FEV1 and MST performance. However, a strong relationship (MST vs FEV1% predicted: r≥0.74, P<0.01 for men and r=0.79, P<0.01 for women) was found below an FEV1 of 67% predicted. CONCLUSIONS: This study suggests that a strong association exists between lung function (FEV1% predicted) and MST (walking distance) in adult patients with moderate-to-severe CF (FEV1<67% predicted). A reference equation for MST performance was developed for those patients with FEV1 ≤67% predicted, providing a tool to make an a-priori prediction of MST walking distance.

CFTR biomarkers: time for promotion to surrogate end-point.

In patients with cystic fibrosis, cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers, such as sweat chloride concentration and/or nasal potential difference, are used as end-points of efficacy in phase-III clinical trials with the disease modifying drugs ivacaftor (VX-770), VX809 and ataluren. The aim of this project was to review the literature on reliability, validity and responsiveness of nasal potential difference, sweat chloride and intestinal current measurement in patients with cystic fibrosis. Data on clinimetric properties were collected for each biomarker and reviewed by an international team of experts. Data on reliability, validity and responsiveness were tabulated. In addition, narrative answers to four key questions were discussed and agreed by the team of experts. The data collected demonstrated the reliability, validity and responsiveness of nasal potential difference. Fewer data were found on reliability of sweat chloride concentration; however, validity and responsiveness were demonstrated. Validity was demonstrated for intestinal current measurement, but further information is required on reliability and responsiveness. For all three end-points, normal values were collected and further research requirements were proposed. This body of work adds useful information to support the promotion of CFTR biomarkers to surrogate end-points and to guide further research in the area.

New clinical diagnostic procedures for cystic fibrosis in Europe.

In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.

Assessment of CFTR function in homozygous R117H-7T subjects.

BACKGROUND: R117H is a frequent missense mutation included in most CFTR mutation panels. However knowledge about the residual function of R117H-CFTR channels in cystic fibrosis-affected organs, e.g. airways, intestines and sweat glands is presently lacking. METHODS: We evaluated clinical CF symptoms and assessed CFTR function by sweat tests, nasal potential difference and intestinal current measurements in 2 homozygous R117H individuals (7T variant). RESULTS: The CFTR activity in airways and intestine was within the normal range. However both individuals presented with a borderline sweat test and the male patient was infertile. CONCLUSIONS: The lack of impact of the R117H mutation on chloride secretion in intestine and nose contrasts with the ~80% loss of CFTR activity reported in patch clamp studies. Apparently CFTR activity is not rate-limiting for chloride secretion in both tissues at levels >20% of normal, or compensatory factors may operate that are absent in heterologous host cells in vitro.

Transient correction of the basic defect in sweat glands in an individual with cystic fibrosis carrying the complex CFTR allele F508del-R553Q.

The molecular pathology of mutant F508del CFTR is partially corrected in vitro by the secondary amino acid substitution R553Q in the ABC signature motif. An individual with the CFTR genotype R553X/F508del-R553Q showed the typical symptoms and electrophysiological anomalies of cystic fibrosis in the airways and intestine. Sweat chloride concentrations were normal early in life, but were later raised into the range that is diagnostic for cystic fibrosis, suggesting that R553Q could temporarily correct the basic defect in sweat glands. R553Q caused a delay in diagnosis because of false negative sweat tests but was not a disease reverting suppressor mutation as had been inferred from cellular models.

Diversity of the basic defect of homozygous CFTR mutation genotypes in humans.

BACKGROUND: Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases. METHODS: 17 individuals who are homozygous for deletions, missense, stop or splice site mutations in the CFTR gene were investigated for clinical symptoms of CF and assessed in CFTR function by sweat test, nasal potential difference and intestinal current measurement. RESULTS: CFTR activity in sweat gland, upper airways and distal intestine was normal for homozygous carriers of G314E or L997F and in the range of F508del homozygotes for homozygous carriers of E92K, W1098L, R553X, R1162X, CFTRdele2(ins186) or CFTRdele2,3(21 kb). Homozygotes for M1101K, 1898+3 A-G or 3849+10 kb C-T were not consistent CF or non-CF in the three bioassays. 14 individuals exhibited some chloride conductance in the airways and/or in the intestine which was identified by the differential response to cAMP and DIDS as being caused by CFTR or at least two other chloride conductances. DISCUSSION: CFTR mutations may lead to unusual electrophysiological or clinical manifestations. In vivo and ex vivo functional assessment of CFTR function and in-depth clinical examination of the index cases are indicated to classify yet uncharacterised CFTR mutations as either disease-causing lesions, risk factors, modifiers or neutral variants.

Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings.

To investigate the impact of chloride (Cl(-)) permeability, mediated by residual activity of the cystic fibrosis transmembrane conductance regulator (CFTR) or by other Cl(-) channels, on the manifestations of cystic fibrosis (CF), we determined Cl(-) transport properties of the respiratory and intestinal tracts in Delta F508 homozygous twins and siblings. In the majority of patients, cAMP and/or Ca(2+)-regulated Cl(-) conductance was detected in the airways and intestine. Our finding of cAMP-mediated Cl(-) conductance suggests that, in vivo, at least some Delta F508 CFTR can reach the plasma membrane and affect Cl(-) permeability. In respiratory tissue, the expression of basal CFTR-mediated Cl(-) conductance, demonstrated by 30% of Delta F508 homozygotes, was identified as a positive predictor of milder CF disease. In intestinal tissue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive (DIDS-insensitive) Cl(-) secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive Cl(-) secretion was observed mainly in more severely affected patients. The more concordant Cl(-) secretory patterns within monozygous twins compared with dizygous pairs imply that genes other than CFTR significantly influence the manifestation of the basic defect.

Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings with cystic fibrosis. The European CF Twin and Sibling Study Consortium.

BACKGROUND & AIMS: Cholinergic stimulation of chloride secretion is impaired in the intestines of patients with cystic fibrosis (CF). However, intestinal chloride secretion has been observed in patients with mild CF mutations. The aim of this study was to investigate residual Cl(-) secretion in the intestine of DeltaF508 homozygous CF patients, and examine the contribution of cystic fibrosis transmembrane conductance regulator (CFTR) and alternative Cl(-) conductances. Twins and siblings with identical CFTR genotypes were investigated to determine the impact of factors other than CFTR on chloride secretion. METHODS: Chloride secretion in rectal tissue was investigated by applying Ca(2+) and adenosine 3',5'-cyclic monophosphate (cAMP)-linked agonists before and after the inhibition of alternative Cl(-) conductances with 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS). RESULTS: cAMP-mediated Cl(-) secretion was observed in 73% of patients, and 20% showed DIDS-sensitive Ca(2+)-activated Cl(-) secretion. This DIDS-sensitive alternative chloride conductance was seen only in CF patients who also responded to cAMP agonists. Chloride secretion was more concordant within monozygous twins than within dizygous pairs. CONCLUSIONS: These results suggest the presence of CFTR-mediated Cl(-) secretion in a subgroup of patients, implying that a portion of deltaF508 CFTR can be processed in vivo and function as a chloride channel in the apical membrane of intestinal cells. Moreover, a considerable number of deltaF508 homozygous patients express chloride conductances other than CFTR in their intestinal epithelia.

Categories of deltaF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics.

Cystic fibrosis (CF), the most common severe autosomal recessive trait among Caucasians, is caused by molecular lesions in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The course of the multi-organ disease CF is highly variable, suggesting the influence of environmental factors and/or modulating genes other than CFTR on the disease phenotype. To evaluate the cause of CF disease variability, the European CF Twin and Sibling Study collected data on two clinical parameters most sensitive for the course and prognosis of CF, ie weight predicted for height (wfh)% (representative for the nutritional status) and FEVPerc (representative for the pulmonary status) for a cohort of 277 sibling pairs, 12 pairs of dizygous twins and 29 pairs of monozygous twins. Of these 318 CF twin and sib pairs, 114 were reported to be homozygous for the most frequent CF disease-causing lesion, deltaF508. Intra-pair discordance was assessed by the intra-pair differences with wfh% and FEVPerc and by DELTA, a composite parameter defined by linear combination of wfh% and FEVPerc in order to describe discordance with respect to the overall disease severity. Monozygous twins had a significantly lower DELTA than dizygous twins (P = 0.05) indicating that CF disease severity is modulated by an inherited component in addition to the CFTR gene itself. Extreme phenotypes are considered to be more informative for the analysis of any quantitative trait. Thus, we aimed to quantify disease severity and intra-pair discordance in order to select pairs with the extreme phenotypes DIS (discordant patient pairs), CON+ (concordant and mildy affected patient pairs) and CON- (concordant and severely affected patient pairs). The algorithm reliably discriminated between pairs DIS, CON+ and CON- among the cohort of deltaF508 homozygotes. The selected pairs from these categories demonstrated non-overlapping properties for wfh%, FEVPerc and the intra-pair difference of both parameters.

Clinical presentation of exclusive cystic fibrosis lung disease.

The diagnosis of cystic fibrosis (CF) is based on the occurrence of two mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and on assays that measure the basic defect of abnormal chloride transport in the affected organs. However, in cases of atypical CF not all diagnostic tests may be positive. We present a patient with an atypical CF phenotype in whom the only presenting symptom was severe CF-like lung disease substantiated by an abnormal nasal potential difference. Genetic analysis showed that the patient was a symptomatic heterozygote, which suggests that one lesion in the CFTR gene may be sufficient to cause CF-like lung disease.

Cystic-fibrosis-like disease unrelated to the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is considered to be a monogenic disease caused by molecular lesions within the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is diagnosed by elevated sweat electrolytes. We have investigated the clinical manifestations of cystic fibrosis, CFTR genetics and electrophysiology in a sibpair in which the brother is being treated as having CF, whereas his sister is asymptomatic. The diagnosis of CF in the index patient is based on highly elevated sweat electrolytes in the presence of CF-related pulmonary symptoms. The investigation of chloride conductance in respiratory and intestinal tissue by nasal potential difference and intestinal current measurements, respectively, provides no evidence for CFTR dysfunction in the siblings who share the same CFTR alleles. No molecular lesion has been identified in the CFTR gene of the brother. Findings in the investigated sibpair point to the existence of a CF-like disease with a positive sweat test without CFTR being affected. Other factors influencing sodium or chloride transport are likely to be the cause of the symptoms in the patient described.

Exocrine pancreatic alterations in long-lived surviving cystic fibrosis mice.

We investigated the development of the exocrine pancreas in Cftr-/- mice in comparison with age-matched littermates (Cftr+/+, Cftr+/-) up to 100 d postnatally. Controls were weaned either to mouse chow or a liquid diet; Cftr-/- mice were weaned solely to a liquid diet. Solid-fed control mice gained weight and showed a progressive increase in pancreatic protein, DNA, amylase, lipase, trypsin, and chymotrypsin activities. Liquid-fed control mice showed similar postnatal somatic and pancreatic growth, except that amylase and lipase activities were lower than in the solid-fed controls. Cftr-/- mice exhibited significantly lower body and pancreatic weights than did controls. Pancreatic protein content and enzyme activities (notably amylase and lipase) were consistently lower than in the age-matched litter-mates fed either diet. The reduction in lipase activity in Cftr-/- mice was noted before weaning. We concluded that the liquid diet influenced postnatal exocrine pancreatic development in mice. However, a further reduction in postnatal pancreatic growth and enzymatic activities in the Cftr-/- mice was noted. These alterations could be due to the primary cystic fibrosis defect, although secondary factors, such as malnutrition induced by decreased dietary intake or abnormal absorptive capacity, may be responsible.