The clinical impact of Lumacaftor-Ivacaftor on structural lung disease and lung function in children aged 6-11 with cystic fibrosis in a real-world setting.

BACKGROUND: Data from clinical trials of lumacaftor-ivacaftor (LUM-IVA) demonstrate improvements in lung clearance index (LCI) but not in FEV1 in children with Cystic Fibrosis (CF) aged 6-11 years and homozygous for the Phe508del mutation. It is not known whether LUM/IVA use in children can impact the progression of structural lung disease. We sought to determine the real-world impact of LUM/IVA on lung structure and function in children aged 6-11 years. METHODS: This real-world observational cohort study was conducted across four paediatric sites in Ireland over 24-months using spirometry-controlled CT scores and LCI as primary outcome measures. Children commencing LUM-/IVA as part of routine care were included. CT scans were manually scored with the PRAGMA CF scoring system and analysed using the automated bronchus-artery (BA) method. Secondary outcome measures included rate of change of ppFEV1, nutritional indices and exacerbations requiring hospitalisation. RESULTS: Seventy-one participants were recruited to the study, 31 of whom had spirometry-controlled CT performed at baseline, and after one year and two years of LUM/IVA treatment. At two years there was a reduction from baseline in trapped air scores (0.13 to 0.07, p = 0.016), but an increase from baseline in the % bronchiectasis score (0.84 to 1.23, p = 0.007). There was no change in overall % disease score (2.78 to 2.25, p = 0.138). Airway lumen to pulmonary artery ratios (AlumenA ratio) were abnormal at baseline and worsened over the course of the study. In 28 participants, the mean annual change from baseline LCI2.5 (-0.055 (-0.61 to 0.50), p = 0.85) measurements over two years were not significant. Improvements from baseline in weight (0.10 (0.06 to 0.15, p < 0.0001), height (0.05 (0.02 to 0.09), p = 0.002) and BMI (0.09 (0.03 to 0.15) p = 0.005) z-scores were seen with LUM/IVA treatment. The mean annual change from baseline ppFEV1 (-2.45 (-4.44 to 2.54), p = 0.66) measurements over two years were not significant. CONCLUSION: In a real-world setting, the use of LUM/IVA over two years in children with CF aged 6-11 resulted in improvements in air trapping on CT but worsening in bronchiectasis scores. Our results suggest that LUM/IVA use in this age group improves air trapping but does not prevent progression of bronchiectasis over two years of treatment.

The effect of CFTR modulators on structural lung disease in cystic fibrosis.

Background: Newly developed quantitative chest computed tomography (CT) outcomes designed specifically to assess structural abnormalities related to cystic fibrosis (CF) lung disease are now available. CFTR modulators potentially can reduce some structural lung abnormalities. We aimed to investigate the effect of CFTR modulators on structural lung disease progression using different quantitative CT analysis methods specific for people with CF (PwCF). Methods: PwCF with a gating mutation (Ivacaftor) or two Phe508del alleles (lumacaftor-ivacaftor) provided clinical data and underwent chest CT scans. Chest CTs were performed before and after initiation of CFTR modulator treatment. Structural lung abnormalities on CT were assessed using the Perth Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF), airway-artery dimensions (AA), and CF-CT methods. Lung disease progression (0-3 years) in exposed and matched unexposed subjects was compared using analysis of covariance. To investigate the effect of treatment in early lung disease, subgroup analyses were performed on data of children and adolescents aged <18 years. Results: We included 16 modulator exposed PwCF and 25 unexposed PwCF. Median (range) age at the baseline visit was 12.55 (4.25-36.49) years and 8.34 (3.47-38.29) years, respectively. The change in PRAGMA-CF %Airway disease (-2.88 (-4.46, -1.30), p = 0.001) and %Bronchiectasis extent (-2.07 (-3.13, -1.02), p < 0.001) improved in exposed PwCF compared to unexposed. Subgroup analysis of paediatric data showed that only PRAGMA-CF %Bronchiectasis (-0.88 (-1.70, -0.07), p = 0.035) improved in exposed PwCF compared to unexposed. Conclusion: In this preliminary real-life retrospective study CFTR modulators improve several quantitative CT outcomes. A follow-up study with a large cohort and standardization of CT scanning is needed to confirm our findings.

The efficacy and safety of systemic corticosteroids as first line treatment for granulomatous lymphocytic interstitial lung disease.

BACKGROUND: Granulomatous and lymphocytic interstitial lung disease (gl-ILD) is a major cause of morbidity and mortality among patients with common variable immunodeficiency. Corticosteroids are recommended as first-line treatment for gl-ILD, but evidence for their efficacy is lacking. OBJECTIVES: This study analyzed the effect of high-dose corticosteroids (≥0.3 mg/kg prednisone equivalent) on gl-ILD, measured by high-resolution computed tomography (HRCT) scans, and pulmonary function test (PFT) results. METHODS: Patients who had received high-dose corticosteroids but no other immunosuppressive therapy at the time (n = 56) and who underwent repeated HRCT scanning or PFT (n = 39) during the retrospective and/or prospective phase of the Study of Interstitial Lung Disease in Primary Antibody Deficiency (STILPAD) were included in the analysis. Patients without any immunosuppressive treatment were selected as controls (n = 23). HRCT scans were blinded, randomized, and scored using the Hartman score. Differences between the baseline and follow-up HRCT scans and PFT were analyzed. RESULTS: Treatment with high-dose corticosteroids significantly improved HRCT scores and forced vital capacity. Carbon monoxide diffusion capacity significantly improved in both groups. Of 18 patients, for whom extended follow-up data was available, 13 achieved a long-term, maintenance therapy independent remission. All patients with relapse were retreated with corticosteroids, but only one-fifth of them responded. Two opportunistic infections were found in the corticosteroid treatment group, while overall infection rate was similar between cohorts. CONCLUSIONS: Induction therapy with high-dose corticosteroids improved HRCT scans and PFT results of patients with gl-ILD and achieved long-term remission in 42% of patients. It was not associated with major side effects. Low-dose maintenance therapy provided no benefit and efficacy was poor in relapsing disease.

Airway Disease in Children with Primary Ciliary Dyskinesia: Impact of Ciliary Ultrastructure Defect and Genotype.

Rationale: Primary ciliary dyskinesia (PCD) is characterized by impaired mucociliary clearance, recurrent respiratory infections, progressive airway damage, and obstructive lung disease. Although the association of ciliary ultrastructure defect/genotype with the severity of airflow obstruction has been well characterized, their association with airway abnormalities on chest computed tomography (CT) has been minimally evaluated. Objectives: We sought to delineate the association of ciliary defect class/genotype with chest CT scores in children with PCD. Methods: Cross-sectional analysis of children with PCD (N = 146) enrolled in a prospective multicenter observational study, stratified by defect type: outer dynein arm (ODA), ODA/inner dynein arm (IDA), IDA/microtubular disorganization (MTD), and normal/near normal ultrastructure with associated genotypes. CTs were scored using the MERAGMA-PCD (Melbourne-Rotterdam Annotated Grid Morphometric Analysis for PCD), evaluating airway abnormalities in a hierarchical order: atelectasis, bronchiectasis, bronchial wall thickening, and mucus plugging/tree-in-bud opacities. The volume fraction of each component was expressed as the percentage of total lung volume. The percentage of disease was computed as the sum of all components. Regression analyses were used to describe the association between clinical predictors and CT scores. Results: Acceptable chest CTs were obtained in 141 children (71 male): 57 ODA, 20 ODA/IDA, 40 IDA/MTD, and 24 normal/near normal. The mean (standard deviation) age was 8.5 (4.6) years, forced expiratory volume in 1 second (FEV1) percent predicted was 82.4 (19.5), and %Disease was 4.6 (3.5). Children with IDA/MTD defects had a higher %Disease compared with children with ODA defects (2.71% higher [95% confidence interval (CI), 1.37-4.06; P < 0.001]), driven by higher %Mucus plugging (2.35% higher [1.43-3.26; P < 0.001]). Increasing age, lower body mass index, and lower FEV1 were associated with a higher %Disease (0.23%; 95% CI, 0.11-0.35; P < 0.001 and 0.03%; 95% CI, 0.01-0.04; P = 0.008 and 0.05%; 95% CI, 0.01-0.08; P = 0.011, respectively). Conclusions: Children with IDA/MTD defects had significantly greater airway disease on CT, primarily mucus plugging, compared with children with ODA defects.

Chest computed tomography outcomes in a randomized clinical trial in cystic fibrosis: Lessons learned from the first ataluren phase 3 study.

A phase 3 randomized double blind controlled, trial in 238 people with cystic fibrosis (CF) and at least one nonsense mutation (nmCF) investigated the effect of ataluren on FEV1. The study was of 48 weeks duration and failed to meet its primary endpoint. Unexpectedly, while FEV1 declined, chest computed tomography (CT) scores using the Brody-II score as secondary outcome measures did not show progression in the placebo group. Based on this observation it was concluded that the role of CT scans in CF randomized clinical trials was limited. However, more sensitive scoring systems were developed over the last decade warranting a reanalysis of this unique dataset. The aim of our study was to reanalyse all chest CT scans, obtained in the ataluren phase 3 study, using 2 independent scoring systems to characterize structural lung disease in this cohort and to compare progression of structural lung disease over the 48 weeks between treatment arms. 391 study CT scans from 210 patients were reanalysed in random order by 2 independent observers using the CF-CT and Perth-Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF) scoring systems. CF-CT and PRAGMA-CF subscores were expressed as %maximal score and %total lung volume, respectively. PRAGMA-CF subscores %Disease (p = 0.008) and %Mucus Plugging (p = 0.029) progressed over 48 weeks. CF-CT subscores did not show progression. There was no difference in progression of structural lung disease between treatment arm and placebo independent of tobramycin use. PRAGMA-CF Chest CT scores can be used as an outcome measure to study the effect of potential disease modifying drugs in CF on lung structure.

Early markers of cystic fibrosis structural lung disease: follow-up of the ACFBAL cohort.

Little is known about early predictors of later cystic fibrosis (CF) structural lung disease. This study examined early predictors of progressive structural lung abnormalities in children who completed the Australasian CF Bronchoalveolar Lavage (ACFBAL) clinical trial at age 5-years and participated in an observational follow-up study (CF-FAB).Eight Australian and New Zealand CF centres participated in CF-FAB and provided follow-up chest computed-tomography (CT) scans for children who had completed the ACFBAL study with baseline scans at age 5-years. CT scans were annotated using PRAGMA-CF scoring. Ordinal regression analysis and linear regression were used to investigate associations between PRAGMA-CF (Perth-Rotterdam Annotated Grid Morphometric Analysis for CF) outcomes at follow-up and variables measured during the ACFBAL study.99 out of 157 ACFBAL children (mean±sd age 13±1.5 years) participated in the CF-FAB study. The probability of bronchiectasis at follow-up increased with airway disease severity on the baseline CT scan. In multiple regression (retaining factors at p<0.05) the extent of bronchiectasis at follow-up was associated with baseline atelectasis (OR 7.2, 95% CI 2.4-22; p≤ 0.001), bronchoalveolar lavage (BAL) log2 interleukin (IL)-8 (OR 1.2, 95% CI 1.05-1.5; p=0.010) and body mass index z-score (OR 0.49, 95% CI 0.24-1.00; p=0.05) at age 5 years. Percentage trapped air at follow-up was associated with BAL log2 IL-8 (coefficient 1.3, 95% CI 0.57-2.1; p<0.001) at age 5 years.The extent of airway disease, atelectasis, airway inflammation and poor nutritional status in early childhood are risk factors for progressive structural lung disease in adolescence.

Multicentre chest computed tomography standardisation in children and adolescents with cystic fibrosis: the way forward.

Progressive cystic fibrosis (CF) lung disease is the main cause of mortality in CF patients. CF lung disease starts in early childhood. With current standards of care, respiratory function remains largely normal in children and more sensitive outcome measures are needed to monitor early CF lung disease. Chest CT is currently the most sensitive imaging modality to monitor pulmonary structural changes in children and adolescents with CF. To quantify structural lung disease reliably among multiple centres, standardisation of chest CT protocols is needed. SCIFI CF (Standardised Chest Imaging Framework for Interventions and Personalised Medicine in CF) was founded to characterise chest CT image quality and radiation doses among 16 participating European CF centres in 10 different countries. We aimed to optimise CT protocols in children and adolescents among several CF centres. A large variety was found in CT protocols, image quality and radiation dose usage among the centres. However, the performance of all CT scanners was found to be very similar, when taking spatial resolution and radiation dose into account. We conclude that multicentre standardisation of chest CT in children and adolescents with CF can be achieved for future clinical trials.

Morphometric Analysis of Explant Lungs in Cystic Fibrosis.

RATIONALE: After repeated cycles of lung infection and inflammation, patients with cystic fibrosis (CF) evolve to respiratory insufficiency. Although histology and imaging have provided descriptive information, a thorough morphometric analysis of end-stage CF lung disease is lacking. OBJECTIVES: To quantify the involvement of small and large airways in end-stage CF. METHODS: Multidetector computed tomography (MDCT) and micro-CT were applied to 11 air-inflated CF explanted lungs and 7 control lungs to measure, count, and describe the airway and parenchymal abnormalities in end-stage CF lungs. Selected abnormalities were further investigated with thin section histology. MEASUREMENTS AND MAIN RESULTS: On MDCT, CF explanted lungs showed an increased median (interquartile range) number (631 [511-710] vs. 344 [277-349]; P = 0.003) and size of visible airways (cumulative airway diameter 217 cm [209-250] vs. 91 cm [80-105]; P < 0.001) compared with controls. Airway obstruction was seen, starting from generation 6 and increasing to 40 to 50% of airways from generation 9 onward. Micro-CT showed that the total number of terminal bronchioles was decreased (2.9/ml [2.6-4.4] vs. 5.3/ml [4.8-5.7]; P < 0.001); 49% were obstructed, and the cross-sectional area of the open terminal bronchioles was reduced (0.093 mm(2) [0.084-0.123] vs. 0.179 mm(2) [0.140-0.196]; P < 0.001). On micro-CT, 41% of the obstructed airways reopened more distally. This remodeling was confirmed on histological analysis. Parenchymal changes were also seen, mostly in a patchy and peribronchiolar distribution. CONCLUSIONS: Extensive changes of dilatation and obstruction in nearly all airway generations were observed in end-stage CF lung disease.

Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells.

Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences between domains are enriched for binding sites of CTCC-binding factor (CTCF) and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on higher-order chromatin organization, as well as the transcriptome. We observed a general loss of local chromatin interactions upon disruption of cohesin, but the topological domains remain intact. However, we found that depletion of CTCF not only reduced intradomain interactions but also increased interdomain interactions. Furthermore, distinct groups of genes become misregulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute differentially to chromatin organization and gene regulation.

In vivo live imaging of RNA polymerase II transcription factories in primary cells.

Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.

Super-resolution imaging reveals three-dimensional folding dynamics of the ß-globin locus upon gene activation.

The chromatin architecture is constantly changing because of cellular processes such as proliferation, differentiation and changes in the expression profile during gene activation or silencing. Unravelling the changes that occur in the chromatin structure during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of an active looping mechanism. It was also shown for the ß-globin locus that the gene promoter interacts with an active chromatin hub by means of an active looping mechanism. This means that the locus changes in three-dimensional (3D) nuclear volume and chromatin shape. As a means of visualizing and measuring these dynamic changes in chromatin structure of the ß-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analysed, and results prior to and after gene activation were quantitatively compared. Confocal and super-resolution imaging revealed that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first non-biochemical evidence that, upon gene activation, an actively transcribing chromatin hub is formed by means of additional chromatin looping.

Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases.

We describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified by both asymmetric and symmetric arginine methylation in vivo. Furthermore, Fop plays an important role in the ligand-dependent activation of estrogen receptor target genes, including TFF1 (pS2). Fop depletion results in an almost complete block of estradiol-induced promoter occupancy by the estrogen receptor. Our data indicate that Fop recruitment to the promoter is an early critical event in the activation of estradiol-dependent transcription.

Stochastic patterns in globin gene expression are established prior to transcriptional activation and are clonally inherited.

We have undertaken a detailed characterization of mouse globin gene expression patterns in the nucleus and cytoplasm of single erythroid cells. We demonstrate an imbalance of alpha- versus beta-globin expression in a significant proportion of cells both in nuclear transcription patterns and cytoplasmic mRNA levels. Clonal cell analysis showed these expression patterns to be clonally inherited, while analysis of a multicopy transgenic locus showed an all-or-none effect in the activation of all the genes in one locus. These data provide strong evidence for a stochastic basis of globin gene activation resulting in heritable all-or-none expression patterns.