Novel Application of Aptamer Proteomic Analysis in Cystic Fibrosis Bronchoalveolar Lavage Fluid.

PURPOSE: Biomarkers are needed in cystic fibrosis (CF) to understand disease progression, assess response to therapy, and enrich enrollment for clinical trials. Aptamer-based proteomics have proven useful in blood samples. The aim is to evaluate proteins in bronchoalveolar lavage fluid (BALF) in CF children compared to controls and identify endotypes during CF exacerbations. EXPERIMENTAL DESIGN: BALF is collected clinically from 50 patients with CF and nine disease controls, processed, and stored per protocol. BALF supernatants are analyzed for 1129 proteins by aptamer approach (SOMAscan proteomics platform). Proteins are compared across groups and used for pathway analysis. Endotypes are identified within the CF group. RESULTS: CF BALF has increased concentrations of neutrophil elastase, myeloperoxidase, and decreased concentration of protein folding and host defense proteins. Pathways that distinguished CF subjects included interferon gamma signaling, membrane trafficking, and phospholipid metabolism. In the CF group, unbiased analysis of proteins identified two distinct endotypes that differed based on BALF white blood cell and neutrophil counts and detection of CF pathogens. CONCLUSIONS AND CLINICAL RELEVANCE: Proteomic analysis of the CF airway demonstrates a complex environment of proteins and pathways. This work provides evidence that aptamer-based proteomics can differentiate between groups and can determine endotypes within CF.

Characteristics and outcomes of oral antibiotic treated pulmonary exacerbations in children with cystic fibrosis.

BACKGROUND: Pulmonary exacerbations (PEx) in children with cystic fibrosis (CF) are frequently treated in the outpatient setting with oral antibiotics. However, little is known about the characteristics of PEx managed on an outpatient basis and the effectiveness of oral antibiotic therapy. We sought to prospectively evaluate clinical and laboratory changes associated with oral antibiotic treatment for PEx. METHODS: Children with CF between 8 and 18 years of age prescribed two weeks of oral antibiotics for a PEx were eligible to enroll. The study consisted of a visit within 48 h of starting antibiotics and a second visit within one week of antibiotic completion. Twenty-eight participants were evaluated by exacerbation score, quality of life measurements, lung function, sputum microbiology and inflammation. RESULTS: Oral antibiotic treatment was associated with a significant improvement in exacerbation score and quality of life measured by the CF Questionnaire-Revised (CFQ-R) respiratory domain. Following treatment, forced expiratory volume in 1 s (FEV1) % predicted increased [median (range)] 9% (-8%, 31%), and 22 (81%) subjects returned to 90% or higher of baseline FEV1. Bacterial density of the primary organism identified on sputum culture decreased significantly with a median (range) decrease of 0.8 log10 cfu/mL (-8 log10, 2 log10, p = 0.03). Sputum neutrophil elastase [-37 µg/mL (-464, 272), p = 0.02] and IL-1ß [-2.8 × 103µg/mL (-6.9 × 104, 3.3 × 104), p = 0.03] decreased significantly following treatment in this cohort. CONCLUSIONS: Treatment of PEx with oral antibiotics was associated with measurable improvements in patient reported outcomes, lung function, bacterial density and sputum inflammatory markers.

Azithromycin for Early Pseudomonas Infection in Cystic Fibrosis. The OPTIMIZE Randomized Trial.

RATIONALE: New isolation of Pseudomonas aeruginosa (Pa) is generally treated with inhaled antipseudomonal antibiotics such as tobramycin inhalation solution (TIS). A therapeutic approach that complements traditional antimicrobial therapy by reducing the risk of pulmonary exacerbation and inflammation may ultimately prolong the time to Pa recurrence. OBJECTIVES: To test the hypothesis that the addition of azithromycin to TIS in children with cystic fibrosis and early Pa decreases the risk of pulmonary exacerbation and prolongs the time to Pa recurrence. METHODS: The OPTIMIZE (Optimizing Treatment for Early Pseudomonas aeruginosa Infection in Cystic Fibrosis) trial was a multicenter, double-blind, randomized, placebo-controlled, 18-month trial in children with CF, 6 months to 18 years of age, with early Pa. Azithromycin or placebo was given 3× weekly with standardized TIS. MEASUREMENTS AND MAIN RESULTS: The primary endpoint was the time to pulmonary exacerbation requiring antibiotics and the secondary endpoint was the time to Pa recurrence, in addition to other clinical and safety outcomes. A total of 221 participants (111 placebo, 110 azithromycin) out of a planned 274 were enrolled. Enrollment was stopped early by the NHLBI because the trial had reached the prespecified interim boundary for efficacy. The risk of pulmonary exacerbation was reduced by 44% in the azithromycin group as compared with the placebo group (hazard ratio, 0.56; 95% confidence interval, 0.37-0.83; P = 0.004). Weight increased by 1.27 kg in the azithromycin group compared with the placebo group (95% confidence interval, 0.01-2.52; P = 0.046). No significant differences were seen in microbiological or other clinical or safety endpoints. CONCLUSIONS: Azithromycin was associated with a significant reduction in the risk of pulmonary exacerbation and a sustained improvement in weight, but had no impact on microbiological outcomes in children with early Pa. Clinical trial registered with clinicaltrials.gov (NCT02054156).

Pulmonary exacerbations and clinical outcomes in a longitudinal cohort of infants and preschool children with cystic fibrosis.

BACKGROUND: Pulmonary exacerbations (PEx) in school aged children and adults with cystic fibrosis (CF) lead to increased morbidity and lung function decline. However, the effect of exacerbations in young children with CF is not fully understood. We sought to characterize the frequency and clinical impact of PEx in a pilot study of infants and pre-school aged children with CF. METHODS: Thirty young children with CF [median (range) 1.5 years (0.2-4.9)] were prospectively followed for 2 years. Exacerbation frequency (hospitalizations and outpatient antibiotic use) was determined. Chest radiographs were performed at enrollment and study completion and assigned a Brasfield score. Lung function at age 7 years was assessed in a subset of children. The association between PEx frequency, chest radiograph score, and lung function was determined using Spearman correlation coefficients and corresponding 95% confidence intervals. Correlations with an absolute magnitude of 0.3 or greater were considered clinically significant. RESULTS: Over 2 years, participants experienced a median of two PEx (range 0-13). Chest radiograph scores at enrollment and study completion were inversely associated with PEx frequency (R = -0.48 and R = -0.44, respectively). The association between frequency of PEx and lung function [forced expiratory volume in 1 s (FEV1)] at age 7 years was small (R = 0.20). Higher forced vital capacity (FVC) at 7 years was associated with more frequent PEx during the study (R = 0.44). CONCLUSIONS: Children with worse chest radiograph scores had more frequent PEx over the subsequent 2 years, suggesting a group of patients at higher risk for PEx. Frequent PEx in infants and young children with CF were not associated with lower FEV1 and FVC at 7 years, although spirometry in this age group may not be a sensitive marker of mild lung disease and disease progression.

Airway microbiota across age and disease spectrum in cystic fibrosis.

Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2 years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ∼50% of the microbiota, whereas in CF patients aged ≥6 years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2 years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.

Early childhood lung function is a stronger predictor of adolescent lung function in cystic fibrosis than early Pseudomonas aeruginosa infection.

OBJECTIVE: Pseudomonas aeruginosa has been suggested as a major determinant of poor pulmonary outcomes in cystic fibrosis (CF), although other factors play a role. Our objective was to investigate the association of early childhood Pseudomonas infection on differences in lung function in adolescence with CF. METHODS: Two populations of subjects with CF were studied: from the Gene Modifier Study (GMS), 346 F508del homozygotes with severe vs. mild adolescent lung disease, and from the Colorado Newborn Screen Study (NBS) 172 subjects diagnosed with CF by newborn screening. Associations of Pseudomonas infection and lung function in early childhood with lung function in adolescence were investigated using multivariate linear regression analyses. RESULTS: Among GMS subjects, those with severe adolescent lung disease had worse lung function in childhood (FEV1 25 percentage points lower) compared to subjects with mild adolescent lung disease, regardless of early childhood Pseudomonas status. Among NBS subjects, those with lowest adolescent lung function had significantly lower early childhood lung function and faster rate of decline in FEV1 than subjects with highest adolescent lung function; early Pseudomonas infection was not associated with rate of FEV1 decline. The strongest predictor of adolescent lung function was early childhood lung function. Subjects with a higher percentage of cultures positive for Pseudomonas before age 6 or a lower BMI at 2-4 years old also had lower adolescent lung function, though these associations were not as strong as with early childhood lung function. CONCLUSIONS: In separate analyses of two distinct populations of subjects with CF, we found a strong correlation between lower lung function in early childhood and adolescence, regardless of early childhood Pseudomonas status. Factors in addition to early Pseudomonas infection have a strong impact on lung function in early childhood in CF. Further exploration may identify novel underlying genetic or environmental factors that predispose children with CF to early loss of lung function.

Proteomic profiling identifies novel circulating markers associated with bronchiectasis in cystic fibrosis.

PURPOSE: Evaluate bronchiectasis change over 1 year in children with cystic fibrosis (CF) and find blood proteins associated with bronchiectasis. EXPERIMENTAL DESIGN: Pilot study of CF children who had chest computed tomography (CT) scans and blood collected during times of clinical stability. Blood plasma was analyzed for 1129 proteins using SOMAmer®, the SOMAscan proteomics platform. Bronchiectasis was measured on two CT scans collected 1 year apart. Spearman's rank estimated the correlations between outcomes. Clinical relevance was defined as |r| >0.40. RESULTS: There were 26 children included: mean age 11.3 years (SD 2.4 years), mean Brody Bronchiectasis score 0.65 (SD 0.83), mean airway count 14.3 (SD 5.7) per CT slice. Brody bronchiectasis change over 1 year ranged from -1.0 to 1.9 and airway count change over one year ranged from -7.7 to 13.5 airways per slice. Proteins related to inflammation and extracellular matrix degradation were associated with cross-sectional and longitudinal structural changes. CONCLUSIONS AND CLINICAL RELEVANCE: Imaging outcomes were more strongly correlated with circulating proteins than age or spirometry values. The unique SOMAscan proteomic platform identifies several novel proteins in blood that are associated with bronchiectasis and that may serve as clinically useful biomarkers in children with CF.

Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function Reduces Airway Bacteria and Inflammation in People with Cystic Fibrosis and Chronic Lung Infections.

RATIONALE: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D-CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established. OBJECTIVES: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections. METHODS: We studied 12 subjects with G551D-CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans. MEASUREMENTS AND MAIN RESULTS: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging. CONCLUSIONS: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D-CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR-targeting treatments.

Diagnosis of Cystic Fibrosis in Screened Populations.

OBJECTIVE: Cystic fibrosis (CF) can be difficult to diagnose, even when newborn screening (NBS) tests yield positive results. This challenge is exacerbated by the multitude of NBS protocols, misunderstandings about screening vs diagnostic tests, and the lack of guidelines for presumptive diagnoses. There is also confusion regarding the designation of age at diagnosis. STUDY DESIGN: To improve diagnosis and achieve standardization in definitions worldwide, the CF Foundation convened a committee of 32 experts with a mission to develop clear and actionable consensus guidelines on diagnosis of CF with an emphasis on screened populations, especially the newborn population. A comprehensive literature review was performed with emphasis on relevant articles published during the past decade. RESULTS: After reviewing the common screening protocols and outcome scenarios, 14 of 27 consensus statements were drafted that apply to screened populations. These were approved by 80% or more of the participants. CONCLUSIONS: It is recommended that all diagnoses be established by demonstrating dysfunction of the CF transmembrane conductance regulator (CFTR) channel, initially with a sweat chloride test and, when needed, potentially with newer methods assessing membrane transport directly, such as intestinal current measurements. Even in babies with 2 CF-causing mutations detected via NBS, diagnosis must be confirmed by demonstrating CFTR dysfunction. The committee also recommends that the latest classifications identified in the Clinical and Functional Translation of CFTR project [http://www.cftr2.org/index.php] should be used to aid with CF diagnosis. Finally, to avoid delays in treatment, we provide guidelines for presumptive diagnoses and recommend how to determine the age of diagnosis.

Diagnosis of Cystic Fibrosis: Consensus Guidelines from the Cystic Fibrosis Foundation.

OBJECTIVE: Cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, continues to present diagnostic challenges. Newborn screening and an evolving understanding of CF genetics have prompted a reconsideration of the diagnosis criteria. STUDY DESIGN: To improve diagnosis and achieve standardized definitions worldwide, the CF Foundation convened a committee of 32 experts in CF diagnosis from 9 countries to develop clear and actionable consensus guidelines on the diagnosis of CF and to clarify diagnostic criteria and terminology for other disorders associated with CFTR mutations. An a priori threshold of ≥80% affirmative votes was required for acceptance of each recommendation statement. RESULTS: After reviewing relevant literature, the committee convened to review evidence and cases. Following the conference, consensus statements were developed by an executive subcommittee. The entire consensus committee voted and approved 27 of 28 statements, 7 of which needed revisions and a second round of voting. CONCLUSIONS: It is recommended that diagnoses associated with CFTR mutations in all individuals, from newborn to adult, be established by evaluation of CFTR function with a sweat chloride test. The latest mutation classifications annotated in the Clinical and Functional Translation of CFTR project (http://www.cftr2.org/index.php) should be used to aid in diagnosis. Newborns with a high immunoreactive trypsinogen level and inconclusive CFTR functional and genetic testing may be designated CFTR-related metabolic syndrome or CF screen positive, inconclusive diagnosis; these terms are now merged and equivalent, and CFTR-related metabolic syndrome/CF screen positive, inconclusive diagnosis may be used. International Statistical Classification of Diseases and Related Health Problems, 10th Revision codes for use in diagnoses associated with CFTR mutations are included.

Prevalence of elevated liver enzymes in children with cystic fibrosis diagnosed by newborn screen.

BACKGROUND: Prevalence and risks for elevated liver enzymes have not been studied systematically in children with CF identified by newborn screen. METHODS: 298 CF children identified by newborn screen since 1982. AST, ALT and GGT tested at annual visits. Percent of children with 1 or ≥2 values of elevated AST, ALT and GGT determined. Relationship of liver enzymes to clinical factors or subsequent liver disease was analyzed RESULTS: At least one abnormal value for AST (63%), ALT (93%) and ALT ≥1.5× ULN (52%) occurred by 21years of age. Liver enzyme elevations were not correlated with CFTR mutation, meconium ileus or ethnicity. AST and GGT ≥1.5× ULN were associated with later advanced liver disease HR (CI) 6.53 (2.02-21.1) and 4.03 (1.15-13.45), respectively. CONCLUSIONS: Elevated liver enzymes are common during childhood in CF patients identified by newborn screen. Elevated AST and GGT may be markers for risk of advanced liver disease.

Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis.

BACKGROUND: Staphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections. METHODS: We examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus directly from airway samples was evaluated. DNA extraction was performed with and without prior enzymatic digestion. RESULTS: 87 samples [42 oropharyngeal (OP) and 45 expectorated sputum (ES)] were analyzed. 59 samples (68%) cultured positive for S. aureus. Using standard extraction techniques, sequencing had the highest sensitivity for S. aureus detection (85%), followed by FemA qPCR (52%) and 16SrRNA qPCR (34%). For all assays, sensitivity was higher from ES samples compared to OP swabs. Specificity of the qPCR assays was 100%, but 21.4% for sequencing due to detection of S. aureus in low relative abundance from culture negative samples. Enzymatic digestion increased the sensitivity of qPCR assays, particularly for OP swabs. CONCLUSION: Sequencing had a high sensitivity for S. aureus, but low specificity. While femA qPCR had higher sensitivity than 16S qPCR for detection of S. aureus, neither assay was as sensitive as sequencing. The significance of S. aureus detection with low relative abundance by sequencing in culture-negative specimens is not clear.

Biomarkers for cystic fibrosis drug development.

PURPOSE: To provide a review of the status of biomarkers in cystic fibrosis drug development, including regulatory definitions and considerations, a summary of biomarkers in current use with supportive data, current gaps, and future needs. METHODS: Biomarkers are considered across several areas of CF drug development, including cystic fibrosis transmembrane conductance regulator modulation, infection, and inflammation. RESULTS: Sweat chloride, nasal potential difference, and intestinal current measurements have been standardized and examined in the context of multicenter trials to quantify CFTR function. Detection and quantification of pathogenic bacteria in CF respiratory cultures (e.g.: Pseudomonas aeruginosa) are commonly used in early phase antimicrobial clinical trials, and to monitor safety of therapeutic interventions. Sputum (e.g.: neutrophil elastase, myeloperoxidase, calprotectin) and blood biomarkers (e.g.: C reactive protein, calprotectin, serum amyloid A) have had variable success in detecting response to inflammatory treatments. CONCLUSIONS: Biomarkers are used throughout the drug development process in CF, and many have been used in early phase clinical trials to provide proof of concept, detect drug bioactivity, and inform dosing for later-phase studies. Advances in the precision of current biomarkers, and the identification of new biomarkers with 'omics-based technologies, are needed to accelerate CF drug development.

Genome-wide association meta-analysis identifies five modifier loci of lung disease severity in cystic fibrosis.

The identification of small molecules that target specific CFTR variants has ushered in a new era of treatment for cystic fibrosis (CF), yet optimal, individualized treatment of CF will require identification and targeting of disease modifiers. Here we use genome-wide association analysis to identify genetic modifiers of CF lung disease, the primary cause of mortality. Meta-analysis of 6,365 CF patients identifies five loci that display significant association with variation in lung disease. Regions on chr3q29 (MUC4/MUC20; P=3.3 × 10(-11)), chr5p15.3 (SLC9A3; P=6.8 × 10(-12)), chr6p21.3 (HLA Class II; P=1.2 × 10(-8)) and chrXq22-q23 (AGTR2/SLC6A14; P=1.8 × 10(-9)) contain genes of high biological relevance to CF pathophysiology. The fifth locus, on chr11p12-p13 (EHF/APIP; P=1.9 × 10(-10)), was previously shown to be associated with lung disease. These results provide new insights into potential targets for modulating lung disease severity in CF.

Considerations for the Conduct of Clinical Trials with Antiinflammatory Agents in Cystic Fibrosis. A Cystic Fibrosis Foundation Workshop Report.

Inflammation leads to lung destruction and loss of pulmonary function in patients with cystic fibrosis (CF). Drugs that modulate the cystic fibrosis transmembrane conductance regulator (CFTR) have recently been approved. Although the impact of CFTR modulators on sweat chloride and lung function are exciting, they have not yet demonstrated an effect on inflammation. Therefore, CF antiinflammatory drug development must continue. Unfortunately, the lack of clarity with this process has left investigators and industry sponsors frustrated. The Cystic Fibrosis Foundation established a working group in early 2014 to address this issue. There are many inflammatory processes disrupted in CF, and, therefore, there are many potential targets amenable to antiinflammatory therapy. Regardless of a drug's specific mechanism of action, it must ultimately affect the neutrophil or its products to impact CF. The working group concluded that before bringing new antiinflammatory drugs to clinical trial, preclinical safety studies must be conducted in disease-relevant models to assuage safety concerns. Furthermore, although studies of antiinflammatory therapies must first establish safety in adults, subsequent studies must involve children, as they are most likely to reap the most benefit. The working group also recommended that pharmacokinetic-pharmacodynamic studies and early-phase safety studies be performed before proceeding to larger studies of longer duration. In addition, innovative study designs may improve the likelihood of adequately assessing treatment response and mitigating risk before conducting multiyear studies. Learning from past experiences and incorporating this knowledge into new drug development programs will be instrumental in bringing new antiinflammatory therapies to patients.

Use of ibuprofen to assess inflammatory biomarkers in induced sputum: Implications for clinical trials in cystic fibrosis.

BACKGROUND: High-dose ibuprofen (HDI) is a clinically beneficial anti-inflammatory regimen that may be a useful reagent to study induced sputum inflammatory marker changes over short study periods appropriate for early-phase CF clinical trials. METHODS: We conducted a 28-day, open-label, randomized, controlled trial among 72 clinically stable CF subjects (FEV1≥40% predicted) randomized to HDI or routine care that assessed IL-6, IL-8, TNF-α, IL-1-ß, free neutrophil elastase, and white cell counts with differentials change from baseline in induced sputum. RESULTS: IL-6 was the only biomarker with significant within-group change: 0.13 log10 pg/mL mean reduction among ibuprofen-treated subjects (p=0.04); and no change in the control group. IL-6 change between groups was statistically significant (p=0.024). No other inflammatory biomarker differences were observed between groups after 28 days. CONCLUSION: Although we studied only one agent, HDI, these results suggest that one month may be inadequate to assess anti-inflammatory candidates using markers from induced sputum.