REVIEW URRENT C OPINION

Cystic fibrosis in the era of genomic medicine Carlos E. Milla

Purpose of review The field of cystic fibrosis (CF) is changing dramatically as the scientific knowledge accumulated since the cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is being translated into effective therapies to correct the basic defect and provide better disease models and in-depth understanding of the basic mechanisms of disease. Recent findings This review focuses on three main aspects of the recent advances in the field: understanding the lung disease pathophysiology (in particular, the early events that condition its onset), better definition of the complex microbiology of the CF airway, and therapeutic developments. Although the most recently developed therapies, whether approved or under study, do not constitute a definitive cure, the benefit to patients is already becoming clearly apparent. Summary As the field continues to change rapidly and new therapies are being identified, CF has become a paradigm for the application of concepts such as translational medicine, genomic medicine, and personalized care, with measurable clinical benefit for the patients affected by this disease. Keywords CF airway microbiology, CF correctors and potentiator therapies, early lung disease

INTRODUCTION The spring of 2013 will mark the 10th anniversary of the announcement of the completion of the human genome project [1], an international research effort to sequence and map all of the genes (together known as the genome) of humans [2]. It was no coincidence that this occurred on the date of the 50th anniversary of the publication of the landmark study by Watson and Crick, who described DNA’s structure as a double helix [3]. The sequencing of the human genome was enthusiastically welcomed as heralding the dawn of a new era in medicine, where the use of specific genetic knowledge was to inform the delivery of effective healthcare [4]. In parallel to this, the development of multiple other ‘-omic’ fields (e.g. proteomics, metabolomics, and transcriptomics) have led to the ability to generate massive amounts of information at the individual level that could be applied to the detection, understanding, and monitoring of disease states [5]. The field of cystic fibrosis (CF) has certainly benefited from these developments. We have witnessed tremendous advances in the diagnostic strategies and our understanding of the disease pathophysiology, and gained better insight into the microbiology of CF. Perhaps of greater importance, we have also

witnessed the success of the first disease-modifying drugs aimed at the basic defect. This review will attempt to summarize the recent advances in the field that have capitalized on the promise of a new era of ‘genomic medicine’.

LUNG DISEASE PATHOPHYSIOLOGY Although generally infants with CF are considered to be free from any lung disease, a substantial body of evidence has accumulated to suggest that the pulmonary manifestations of the disease have a very early onset, if not at birth itself. The earliest systematic descriptions of the lung pathology in young children with CF pointed to focal changes at the small airways level, with the presence of mucus plugging and dilatation of the airway lumen, in addition to hyperplasia of the bronchial glands [6]. Several studies conducted in asymptomatic Correspondence to Carlos E. Milla, MD, Department of Pediatrics, Center for Excellence in Pulmonary Biology, Stanford University, 770 Welch Road, Suite 350, Palo Alto, CA 94304, USA. Tel: +1 650 736 9824; e-mail: [email protected] Curr Opin Pediatr 2013, 25:323–328 DOI:10.1097/MOP.0b013e328360dbf5

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KEY POINTS  Although structurally normal at birth, an inability to control bacterial infection plays a key role in the early CF lung disease pathogenesis.

chronic infections in CF patients. Additional studies with the CF pigs have also provided insight into another significant respiratory problem in CF patients: sinus disease. Though free from infection and inflammatory changes, the sinus cavities of CF piglets are found to be hypoplastic [20 ]. Infection becomes established later and adult CF pigs present all of the features seen in the human disease. An investigation of the development of chronic Pseudomonas infection in the Danish CF patient cohort provides further insight by demonstrating that the paranasal sinuses constitute a niche for early infection and the source of lower airway infection. Of interest, the investigators also found a number of phenotypic adaptations of the Pseudomonas colonies in the sinuses that preceded the lower airway colonization [21]. Taken together, these two studies provide evidence for an abnormal milieu in the sinuses that provides an initial niche for the evolution of the Pseudomonas infection. Early and subtle small airways abnormalities are unlikely to produce any noticeable symptoms. This disconnect between the presence of pathological changes and symptomatology implies that more sensitive diagnostic methods must be employed to detect the presence of airway disease to clearly identify the presence and cause of the earliest pathologic events. Given the challenges posed by young age and consequent lack of cooperation in this patient population, innovative methodologies have been applied to the study of airway disease in infants with CF. As a result, it is increasingly accepted that with the application of the appropriate techniques it is quite feasible to detail even early lung disease in these infants. Both functional and imaging methodologies have been effectively applied and have yielded important information in longitudinal studies. From a functional perspective, multiple studies have clearly established that infants with CF have lung function abnormalities that result from airway obstruction and that these abnormalities persist in the later years of life [22–25]. More recent studies applying less invasive techniques such as multiple breath washout methodologies with the use of tracer gases have provided further insight into these abnormalities [26]. These studies demonstrate that regional small airway disease results in ventilatory inhomogeneity and this occurs early, well prior to being detectable by forced expiratory maneuvers [27 ,28]. In addition, the presence of Pseudomonas infection is associated with abnormalities in Lung Clearance Index (LCI) [29], a well-validated surrogate measure of ventilatory homogeneity [30]. Findings from the functional studies have been corroborated by structural studies with the use of computerized tomography (CT) imaging. If anything, data available &

 The application of novel molecular techniques to explore the human microbiome has produced paradigm shifting information on the CF airway microbiology.  With the approval of the drug Ivacaftor, the concept of using small molecules as CFTR corrector and potentiator therapies has been proven as a valid strategy to produce meaningful clinical benefit.

infants with CF identified by newborn screening have repeatedly demonstrated the presence of active inflammation and infection in the lower airway [7–12], and their association with progression in the pulmonary disease [13]. The availability of a large animal model of CF lung disease through the generation of transgenic pigs with the disease has deepened our understanding of the earliest events in CF lungs [14,15]. Piglets with CF present at birth with severe bowel obstruction and if rescued from this go on to develop severe lung disease [14,16]. Although no abnormalities are detected in their lungs at birth, shortly after CF pigs develop all the hallmarks of CF lung disease, including mucous accumulation, inflammation, and infection. Interestingly, although both control and CF pigs have bacteria detectable in the lower airway shortly following birth, the CF pigs seem unable to eradicate the bacteria from their lungs, suggesting a strong role for unique host–environment interactions at the genesis of CF lung disease [16]. Then, this animal model supports the notion that the development of lung disease has its onset much earlier than anticipated and likely as a consequence of a failure to eliminate bacteria from the airway. These observations have been further clarified by a recent study with this pig model of CF identifying a defect in the ability of the airway mucosa to eradicate the bacteria [17 ]. Compared with wildtype pigs, airway surface fluid from CF pigs has a lower pH and reduced antimicrobial activity. Further, increasing the pH rescued this defect and restored the antimicrobial activity of the airway surface fluid. Given that the cystic fibrosis transmembrane conductance regulator (CFTR) is already recognized as participating in bicarbonate transport [18,19], the results of this study provide a direct mechanistic explanation for the failure to clear infections known to occur in the CF airway and suggest, perhaps, a potential novel therapeutic intervention to prevent the serious &&

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from several studies seems to indicate that functional and structural studies provide complementary information [31,32]. Perhaps the best body of evidence has come from investigators in Australia, who demonstrated by the use of CT scan imaging methodology the presence of bronchiectasis at an early age in patients identified by newborn screening [33]. A recent follow-up study to their initial observations on this cohort demonstrated the longitudinal progression of these abnormalities [34 ]. Notable findings in this study included not only the persistence and progression of bronchiectasis and air trapping, but also the association of this progression with worsening of infection and neutrophilic inflammation in the lower airway. Whether functional studies could be used to screen those patients in whom additional valuable information could be obtained with imaging studies has not been explored. This is an important question as, despite the refinements in technology, imaging studies still involve radiation exposure. A recent panel convened by the US National Heart, Lung, and Blood Institute to identify priority areas for the study of early CF lung disease highlighted the need to develop novel biomarkers and outcomes measures in order to gain further insight into the pathophysiology of early lung disease and, perhaps more importantly, to identify effective interventions in the youngest patients [35]. &&

CYSTIC FIBROSIS MICROBIOLOGY Despite the major advances in CF care, progressive lung disease in association with chronic infections remains the major cause of morbidity and mortality in CF [36]. It is also clearly recognized that the presence of infection in the airways is a dynamic process that evolves as the patients age [37], likely a product of multiple factors including adaptation of the bacteria to the airway environment and response to the antibiotic therapy [38,39]. Frequently infections become established silently and even if detected they could persist in spite of appropriate application of antibiotic therapy. In addition, even when infections can be clearly demonstrated, it is also recognized that the clinical response to the most appropriate antibiotic regimens cannot be fully predicted and tremendous variability exists from patient to patient [40]. Further, it is a common observation in the clinical setting that antibiotic therapy confers clinically detectable benefit even in the context of continuing infection with antibiotic-resistant organisms [41]. This apparent paradox to the traditional paradigm of infectious diseases wherein pathogenesis derives from a primary and identifiable organism that can be effectively targeted has been difficult to explain.

To reconcile this paradox, a complex multifactorial process has been proposed in which the conditions of the airway environment itself, the host inflammatory response, and the characteristics of the offending microorganisms, including phenotypic changes with reduced expression of virulence genes and adaptive mechanisms, determine the disease severity [42–45]. However, this is still based on a paradigm of a sole causative, or at least dominant, microorganism and ignores the possibility of a more complex microbial community present in the airway. The availability of nonculture-based microbial identification methodologies has opened the possibility of performing a more comprehensive assessment of the microbial ecology in different niches of the human body [46]. Recently, the application of microbiological identification tools based on 16S rRNA gene analysis has revealed a highly complex microbial ecology in the CF lung [47–56]. Additionally, evidence has become available for the presence in the lower airway of non-CF healthy individuals of low levels of bacteria found to be present also in the upper airway [57]. This suggests that bacteria are not only frequently reaching the lower airway, but also effectively cleared, a condition that is known to be defective in CF patients. The assessment of the microbial communities present in the airways of CF patients has been rapidly growing [44]. A recent comprehensive quantitative analysis of the CF pulmonary microbiome revealed the presence of a specific signature not seen in healthy controls [58 ]. The pattern identified revealed not only the predominance of certain microbial families, but also the absence or decreased abundance of several families identified in the healthy controls. Further, a significant association between microbial diversity and clinical parameters, including inflammatory biomarkers, was apparent among the CF patients. Additional studies have expanded on this knowledge by starting to reveal important interactions at the microbial community level, including the presence of fungal species [59], spatial heterogeneity in the distribution of the microbial communities in the lungs [60], loss of community diversity with disease progression [61 ], and close longitudinal relationships in the evolution of the lung and the gut microbiome [62]. Even so, the development of the CF lung microbiome from the time of birth, as well as the local and systemic host responses triggered as the immune system develops, has not been explored fully yet. With the molecular and genomic tools available, there is a great opportunity at hand to understand the composition of microbial communities in the CF airway, how their development influences the progression of lung disease, and how the

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composition of these communities has an effect on the differential responses to therapeutic intervention.

THERAPEUTIC DEVELOPMENTS Perhaps the most remarkable recent advance in CF pertains to the approval of the first drug targeting the basic defect. Ivacaftor (N-[2,4-di-tert-butyl-5hydroxyphenyl]-1,4-dihydro-4oxoquinoline-3-carboxamide) received approval by the Food and Drug Administration in January 2012 for the treatment of CF patients carrying at least one copy of the G551D mutation. The approval of this drug followed from unprecedented efficacy results in short-term [63] and long-term [64 ] clinical trials. The drug is a small molecule identified through high-throughput screening (HTS) with the use of an in-vitro cell reporter system [65]. The application of HTS for drug discovery in CF followed the development of the methodology to screen in cell cultures compounds with activity on ion channels [66]. The ability to screen thousands of chemical compounds rapidly permitted testing the concept that CFTR function can be restored by rescuing mutated CFTR protein from degradation and potentiating its activity in the apical cell membrane [67]. A number of compounds and chemical classes have been identified utilizing this strategy and have validated the use of this approach for drug discovery in CF [68–71]. As a result, at least two additional compounds are currently far along in clinical trials. The drug VX-809 has already been demonstrated in vitro to have activity as a corrector of CFTR with the F508del mutation [72]. Although the F508del mutation is already well demonstrated to affect CFTR folding, thereby preventing its trafficking to the apical membrane, recent data suggests that even if rescued the protein will have decreased activity and perhaps also a low residence time in the membrane. Not only are the thermodynamics of the protein significantly affected by the F508del mutation, but also the interaction of the first nucleotide-binding domain with adjacent transmembrane spanning domains is disrupted, which results in defective transport function [73,74 ,75,76]. Thus, it is not surprising to learn from the results of a recently reported clinical study that, among patients homozygous for F508del treated with VX-809, minimal effects in sweat chloride values and no other significant effects were noted after 28 days of treatment. Then, a combination therapy approach with the use of a corrector such as VX-809 and Ivacaftor as a potentiator has been proposed, and is currently under test in clinical trials. Preliminary results of an ongoing clinical trial in CF patients homozygous for the F508del mutation &&

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have already shown some promising results [77]. A second corrector agent, VX-661, is also being investigated in clinical trials in combination with Ivacaftor as a second corrector–potentiator therapeutic regimen (NCT01531673). The results of these studies are highly anticipated. An additional therapeutic under development targets CFTR mutations that result in a premature termination codon (PTC). The drug Ataluren is being developed as a suppressor of PTC mutations. Early small clinical studies with CF patients carrying at least one copy of a PTC mutation showed promising results, with detectable effects in biomarkers of CFTR function such as the nasal potential difference [78–80]. However, a larger international study with longer-term exposure to the drug did not find a meaningful effect on pulmonary function or exacerbation rate. In addition, the application of in-vitro cell assay platforms has helped to identify other CFTR mutations in which Ivacaftor could potentially have a beneficial effect [81 ,82]. This has also presented the possibility of utilizing in-vitro cell-based assays to identify patients who could potentially benefit from novel CF therapies. Given the low frequency of many mutations known to be associated with CF, this could represent a more efficient approach instead of embarking on the traditional path of large clinical trials, which will be impossible to conduct for rare mutations. In addition to the benefits realized by a small group of patients, the success in the development of Ivacaftor can also be taken as a successful proof of concept. This has demonstrated that the CF basic defect is amenable to targeting for effective restoration of CFTR function and that, as a result, important clinical benefits are realized. As important, the experience acquired during the Ivacaftor clinical trials has also helped identify the utility of physiologic biomarkers, such as sweat chloride and nasal potential difference, in their ability not only to detect meaningful effects, but also to predict clinical benefit. &

CONCLUSION The field of CF is undergoing dramatic and rapid change that takes full advantage of the fast pace at which multiple ‘-omics’ platforms continue to develop. This has produced not only a more detailed understanding of the basic mechanisms of disease, but also the first-in-class drugs directed at the basic defect. Clearly, the basic defect in this disease can be effectively targeted for correction with small molecule drugs, and many of these novel drugs will have a mutation-specific effect. In addition, our Volume 25  Number 3  June 2013

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understanding of the disease pathogenesis has been better informed as novel tools to dissect the basic molecular mechanisms of protein dysfunction and improved animal models have become available. The promise of genomic medicine and personalized care is then being fully realized by patients and families affected by CF. Acknowledgements None. Conflicts of interest There are no conflicts of interest.

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Volume 25  Number 3  June 2013

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