Highlights of the North American Cystic Fibrosis Conference 2011

This year's meeting was held in Anaheim, California, and was an excellent mix of basic science, translational research and clinical practice. For the first time, authors were invited to submit accepted abstracts as electronic posters (http://nacfcdl.cff.org/Pages/E-Posters.aspx). The meeting was heavily dominated by the hot-off-the-press news of the phase 3 trial results of Ivacaftor (VX-770), which is discussed in detail below. Accompanying this was a resurgence of enthusiasm surrounding the potential of CFTR as a therapeutic target, with possible implications for other small molecule approaches and gene therapy.

We have structured this review around the following areas: (a) Genetics, including newborn screening; (b) pathophysiology and animal models; (c) conventional management and monitoring and (d) progress in novel therapies.

Genetics

CFTR2

The Clinical and Functional TRanslation of CFTR (CFTR2) project is an ambitious endeavour to increase understanding of the clinical consequences of an increased number of CFTR mutations. An excellent plenary session was co-presented by Garry Cutting and Patrick Sosnay (Available online with other Plenary sessions at http://www.cff.org/research/NACFC/2011NACFC/). They described how the current panel of 23 mutations recommended by the American College of Medical Genetics, represents only 1.2% of all mutations (over 1,800 in total) reported in the CFTR gene, but accounts for 85% of mutations occurring in CF patients and covers both affected alleles in 72% of patients. The group has collected data from 23 registries/clinics worldwide, comprising over 39,000 patients. Specifics of genotype, sweat chloride, FEV₁% predicted and pancreatic exocrine status are included. The focus of the first phase is on 160 mutations with an allele frequency >0.01% (at least 9 patients on the database). These are being categorised into (a) disease causing mutations, (b) neutral variants (non-CF causing polymorphisms) or (c) of uncertain significance based on clinical (mean sweat chloride for all patients in the group >60 mmol/l), functional (<10% wild type levels on Ussing chamber experiments) and pattern of inheritance considerations (the mutation cannot be present on the alternative allele of fertile obligate heterozygote fathers). Interestingly, several mutations (e.g. S1235R) have been conclusively shown not to be CF-causing, whilst others remain unclear; 126 have been confirmed deleterious. The project is in its infancy but will be extremely useful to those in the field of CF involved in the design and implementation of newborn screening programmes, and the diagnosis of non-classic cases. The website will go live early in 2012 and will be accessible to the professional and lay population alike; feedback and evaluation will be actively sought in this dynamic process.

Newborn Screening

Themes emerging from the NBS abstracts related largely to missed diagnoses and borderline cases. The UK's national newborn screening programme for CF was commenced in July 2007. However, in East Anglia NBS started in 1980 and so 30 years of data are now available (#429).¹ Over 730,000 babies have been screened with 296 CF cases detected; 29 cases (8.9%) were missed. In the 10 of these with meconium ileus (MI), it is thought that surgery, or perhaps lack of feeding, may have affected the immunoreactive trypsinogen (IRT). The remaining 19 missed patients were diagnosed between 4 weeks and 14 years of age; 12 were pancreatic insufficient. There were 91 false positives, half of whom were well term babies and half had other abnormalities such as organ failure or chromosomal trisomies. New Zealand has also been screening infants for 30 years and review of their data revealed 7.2% of cases were missed initially (#433). A study from Georgia, USA, found significantly higher IRT levels in African-American babies than Caucasians (#421) and in babies born weighing <1500 g. In California, following a high IRT, blood samples are sent for full CFTR sequencing; this raises questions relating to the clinical significance of novel or rare mutations. Patients with such mutations with negative sweat tests are followed up and it was reported that their sweat chloride increases with age (#416). It was evident that 25% of them displayed clinical symptoms and were labelled as CF, despite not fulfilling standard diagnostic criteria (#S5.3). In contrast, the Toronto group presented data on babies with an uncertain diagnosis (borderline sweat chloride and either only one mutation or two, but one of which was of uncertain significance). The majority (23/25) carried two CFTR mutations. However, unlike a group of definite CF babies, their sweat chloride decreased over time. Of the 25 uncertain patients, only one has since received a CF diagnosis, based on abnormal sweat chloride. This group conclude that neither symptoms nor the carriage of CF-associated organisms was useful in establishing or ruling out the diagnosis. Both these groups agree however that, in cases of doubt, serial sweat testing is useful.

Pathophysiology

Model systems

Mucociliary clearance is an integral component of innate lung defence, but our understanding remains limited, partly due to technical difficulties in capturing ciliary motion and three-dimensional interactions between cilia and mucus in intact epithelia; scanning electron microscopy (SEM) is destructive to cells and video microscopy lacks sensitivity. 1-μM resolution optical coherence tomography (μOCT) is a technique that produces video-rate, cross-sectional images at the sub-cellular level and demonstrates clear differences between CF and non-CF cultures in mucus transport rate. The technique has demonstrated that normal respiratory epithelial cells (both human bronchial epithelia and swine tracheal tissue) respond dynamically to mucus loads anticipated to occur in CF by increasing ciliary beat frequency. CF epithelia cannot respond in this way, probably as a result of interdigitation of mucus within the cilia preventing increased beat frequency (#71). A respiratory cell culture model (#114) of human sinonasal epithelium studied the stimulation of CFTR-mediated Cl⁻ conductance and ciliary beat frequency by the CFTR potentiating drug, VX-770, using high speed digital imaging, and found that transepithelial Cl⁻ transport and ciliary beat frequency were strongly activated. The magnitude of Cl⁻ secretion did not correlate directly with ciliary beat frequency, suggesting that augmentation may display a threshold effect. These findings suggest that markers of sinonasal mucociliary clearance may be useful outcome measures of CFTR-modulators.

Animal models

Animal model abstracts were numerous but, as transgenic CF mice demonstrate little in the way of lower airway disease, we focus more here on the newer pig and ferret models, which may be more relevant to our understanding of lung pathophysiology.

1. A host defence defect is present at birth in both models, demonstrated by impaired eradication of experimental infection with Pseudomonas aeruginosa by CF ferrets and S. aureus by CF pigs. The mechanism of the innate immune defect is unclear but, at least in the ferret, it is probably independent of impaired mucociliary clearance; it occurs early in life before much of the airway has become ciliated and when first measured, MCC is normal in the CF animals (#74). In CF ferrets, bactericidal activity can be restored by adenovirus-mediated CFTR gene transfer; xenograft experiments suggest that only the surface epithelium required correction, without the need for correction of underlying submucosal glands (#74);

2. CF pigs and ferrets develop spontaneous airway infections;

3. Abnormalities of airways surface liquid (ASL) are thought to contribute to CF lung disease; ASL in the CF pig was shown to have increased viscosity, possibly attributable to reduced pH and/or HCO₃⁻ (#73) although antimicrobial activity was similar to controls (#163);

4. CF pig lung histology at a few months of life demonstrates inflammation and remodelling, although in newborn pigs such histological changes are absent and proinflammatory cytokines are not raised. This suggests infection precedes inflammation.

Microbiology

There is increasing recognition that our conventional view of the lower airway as being ‘sterile’ in health and infected by a ‘narrow range of bacterial pathogens’ in CF is completely incorrect. Several abstracts were presented on the detection of lower airway pathogens by non-culture based molecular techniques (#260–3). Most investigators agree that multiple pathogens are present in the BAL (bronchoalveolar lavage) or sputum and there are some emerging data to suggest that an infective exacerbation is accompanied not by new or increased numbers of organisms, but by shifts in populations and diversity. Once again, the regional variation in CF lungs was demonstrated by distinct genetic subpopulations of Pseudomonas aeruginosa being encountered in different lobes (#249). The prevalence of these subpopulations changed significantly during an infective exacerbation (#250). Sampling from the upper airway may be very misleading (#263). Patrick Flume, in his excellent plenary lecture on infective exacerbations (P3), raised the important question of how we can recognize the harmful pathogens amongst this bacterial ‘soup’; this active area of research needs to address some questions of clinical relevance to fulfil its promise.

Lung defence

There was once again a debate surrounding whether or not there is a primary defect in CF circulating inflammatory cells; as this field evolves, the consensus seems to be ‘Yes’ although the level of such a defect may be small and clinical significance in humans is uncertain (S12.1). There were several abstracts on the potential role of vitamin D in lung innate defence, various groups reporting on the relationship between serum vitamin D level and lung function although results were conflicting. (#S2.2, #484, #144, #83, #496). A systematic review of this topic (#517) concluded that there is a weak positive correlation. A small intervention study randomized 30 patients admitted to hospital with pulmonary exacerbation to IM vitamin D or placebo (#526). They measured lung function, vitamin D and cathelecidin – an antimicrobial peptide thought to be induced by vitamin D. In the vitamin D group, cathelecidin was significantly increased at weeks 1 and 12. It was shown that 78% of patients had a return of their FEV₁ to baseline compared with just 38% of the placebo group, although this failed to reach statistical significance. The CFF established a working party to formulate guidelines for vitamin D levels deficiency (currently in submission)(#S2.2), which recommend annual vitamin D levels, ideally towards the end of winter when vitamin D levels reach their nadir; 25(OH) levels should be used and a level >30 ng/mL (75 mmol/L) is recommended; vitamin D3 (cholecalciferol) is more effective than D2 (ergocalciferol) and should be given orally, either daily or weekly; levels should be re-checked 3 months after commencing supplementation. A dosing plan is given within the guidelines. Vitamin D levels often increase post lung transplantation (#S2.3) without alteration of supplementation. Many drugs used post-transplant affect the metabolic pathway, either reducing or (more commonly) increasing serum vitamin D levels. This highlights the importance of considering drug interactions when commencing new medications.

Conventional management and monitoring

A retrospective evaluation (#330) of 180 courses of intravenous antibiotics in 81 children showed that 47% returned to baseline predicted FEV₁ on Day 7 of IV treatment, compared to 64% by Day 14 (P < 0.005). Median FEV₁ on Day 7 was 77% of predicted, compared with a median of 83% predicted on Day 14 of treatment. An Australian trial on 63 subjects over 5 months failed to find any clinical benefit of the oral macrolide, clarithromycin (#351). An interesting case was presented in the Fellows' session of persistent, severe haemoptysis responding promptly and dramatically to treatment with the beta-blocking agent, atenolol. The mechanism is unclear but likely relates to reduction in pulmonary arterial pressures.

The validity of FEV₁, as a primary outcome measure in CF trials was questioned (S14.4), as the annual change is small and may lead to underestimation of disease severity, although it is still applicable if a large treatment effect is anticipated (as in the case of VX-770). Lung clearance index may be abnormal in children with normal FEV₁, although it may not correlate with bronchiectasis and air trapping on high resolution CT scan (HRCT) in children under the age of two. Although HRCT is sensitive and reliable, and standardised automated interpretation is on the horizon, it is not easily repeatable due to concerns regarding anaesthesia and radiation exposure. An Australian study (#S5.2) has enrolled CF babies diagnosed by NBS and found that by the age of 3 years, 75% had CT scan evidence of bronchiectasis; presence was associated with a severe genotype, pulmonary infection and neutrophilic inflammation. Although pulmonary exacerbations remain difficult to define accurately, data from the Toronto group indicate that they are responsible for half of the decline in lung function observed over a 6–7 year time period; perhaps we should be focusing on them more? It is apparent that the lack of sensitive biomarkers to track disease progression is a significant unmet need in CF. Those discussed at the NACFC included CRP (#81), catecholeamines in airway fluid (#84), procalcitonin (#247), cytokines in CF-related arthropathy (#241) and arylsulfatase B (#233); the reliability and validity of nasal PD was reviewed (#228). There were two novel biomarkers that we felt were worth highlighting: (a) CD203c and CD63: Basophils play a major role in the allergic response and it was shown that their surface levels of CD203c and CD63 increase significantly upon stimulation with Aspergillus fumigatus, a finding that is allergen-specific. CD203c was also found to be an excellent predictor for discriminating between colonized patients and those with allergic bronchopulmonary aspergillosis (ABPA), and for monitoring treatment response; at £200 per assay it may be a viable biomarker in Aspergillus management; (b) CD16b-AAT: CD16b-alpha-1-antitrypsin is a marker of neutrophil priming that is shed when the neutrophil membrane is exposed to chemokines such as IL-8. CD16b-AAT and IL-8 levels correlated and both fell in response to antibiotic treatment (P = 0.03), whilst FEV₁ pre- and post-exacerbation was inversely associated with both biomarkers (#85). Hence, CD16b-AAT may represent a promising biomarker of inflammatory status in CF and, as neutrophil priming must, by definition, occur prior to activation and migration of neutrophils towards the lung, monitoring levels may allow exacerbations to be detected prior to evolution of clinical symptoms.

Progress with novel therapies

Small molecule drugs

Ataluren

This drug, aimed at allowing ribosomal readthrough of nonsense (class 1 mutations) is being studied in a large, multinational phase 3 trial. At the time of the conference, baseline data were available, confirming that this group of 238 patients has a significant burden of disease: at a median age of 23 years, FEV₁ was reduced to 60%, respiratory domain of the CFQ-R quality of life questionnaire to 72/100. Sweat chloride and nasal electrophysiological measurements were typical for classic CF. These data would suggest (a) scope for a treatment effect being detectable on these biomarkers and (b) that this population has significant manifestations of disease which could be improved if the trial confirms the drug is beneficial.

Ivacaftor (VX-770)

The report of the first phase 3 trial of this oral agent appeared in press during the conference, further increasing the impact of the results, which were discussed in plenary, symposia and several workshop presentations. Ivacaftor has been designed to target class 3, so-called ‘gating’ mutations, as a result of which CFTR reaches the cell surface but fails to open. G551D is by far the commonest gating mutation worldwide and to date, it is patients with this mutation who have been included in trials. Bonnie Ramsay presented the phase 3 data from 161 patients aged 12 years and above, treated twice daily for 48 weeks (#211). Primary outcome was change in FEV₁ at 24 weeks; the treatment effect for absolute change was 10.6%, which translated into a relative improvement over baseline of 16.7%. In addition, actively-treated subjects experienced fewer respiratory exacerbations and had significant improvements in the CFQ-R respiratory domain and weight. Sweat chloride levels fell (treatment effect −47.9 mmol/l), many of them to below the CF diagnostic cut-off of 60 mmol/l. There were no safety concerns. In the open label extension study (#204), the previous placebo group demonstrated similar improvements in FEV₁ over the 12 weeks reported. The drug showed comparable, dramatic effects in the phase 3 trial in children aged 6–11 years with G551D (#203) and is showing promise in a phase 2 trial in patients with mild lung impairment using LCI as a more sensitive measure of airway disease than FEV₁ (#201). VX-770 was shown to possess potentiating capability for other gating mutations (#10), although these are all rare in the CF population. The agent had no effect on subjects homozygous for the Phe508del mutation when used alone, but in combination with the corrector agent, VX-809, it led to a significant drop in sweat chloride (#212); further studies are ongoing to assess whether this is of a sufficient magnitude to lead to clinical benefits. The recent applications for marketing approval in both the US and Europe were highlighted in several presentations, raising hopes that this drug may be available in the clinic in the near future.

CFTR correctors

Correctors, drugs which allow misfolded CFTR to traffic to the cell surface, have been long sought after at great expense by academics and the pharmaceutical industry alike. The description at this year's conference of two separate misfolding events in Phe508del (in both NBD1 and domain assembly) perhaps goes some way to explaining why this has proved so difficult to date. However, the success of VX-770 has clearly catalyzed a renewed resurgence of interest in small molecule therapies. The CFF announced a major new collaborative project with Genzyme, investigators highlighted Pfizer's corrector programme and the Vertex compound VX-661 was mentioned.

Antibiotic agents

The results of a multi-cycle open-label trial of nebulized Arikace (liposomal Amikacin) in patients with chronic Pseudomonas aeruginosa infection, showed that FEV₁ had increased by 11.7% at the end of treatment with a sustained increase of 5.7% after 56 days off 6^th cycle of treatment. There was a significant reduction in density of Pseudomonas aeruginosa and no significant shift in the mean inhibitory concentration (MIC). Phase 3 studies are underway (#221). A placebo-controlled, double-blind randomized controlled trial of Aztreonam for inhalation (AZLI, #234) found no difference in FEV₁ or any secondary outcome measure in 100 patients chronically infected with Burkholderia cepacia complex. AZLI was compared to Tobramycin inhalation solution (TIS) in patients with chronic Pseudomonas aeruginosa with significantly more patients on AZLI displaying either ≥8% or ≥12% improvement in FEV₁ at 28 days, an effect that persisted over three treatment courses (#216). 288 patients chronically infected with Pseudomonas aeruginosa have been randomized to Ciprofloxacin dry powder for inhalation (DPI) or placebo in a Phase IIB study and data analysis is ongoing (#235).

Gene therapy

We presented data from the UK CF Gene Therapy Consortium from both animal toxicology (#238, #197) and human (#198) studies. The non-viral formulation is safe in repeated dose animal studies and encouragingly, shows increased expression with repeated dosing. A safe dose was established from the single dose human pilot study, with long duration of functional efficacy being demonstrated in some subjects on nasal electrophysiological testing. Funding permitting, a repeated dose trial designed to assess clinical benefit will begin in early 2012.

Summary

The conference succeeded once again in bringing together basic scientists, clinicians and the allied professions in a rich mix of presentations. The additional excitement generated by the success of a new drug heightened the usual levels of enthusiasm. To have, for the first time, efficacy with a small molecule is worth so much more than the hope it provides to the 1 in 25 CF patients fortunate enough to possess a Class 3 mutation. It tells us that CFTR is a molecule worth targeting; the effort may be huge, but the rewards are potentially even more so.

DECLARATIONS

Competing interests

JCD is Principal Investigator on current trials sponsored by Vertex and PTC Pharmaceuticals and has served on Advisory Boards for Vertex, PTC, Novartis, Forest and Bayer

Funding

JCD is funded by the CF Trust via the UK CF Gene Therapy Consortium

Ethical approval

Not applicable

Guarantor

Not applicable

Contributorship

All authors attended the conference and contributed to all drafts of this manuscript

Acknowledgements

None