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Inhaled antibiotics are commonly used to treat persistent airway infection with Pseudomonas aeruginosa that contributes to lung damage in people with cystic fibrosis. Current guidelines recommend inhaled tobramycin for individuals with cystic fibrosis and persistent Pseudomonas aeruginosa infection who are aged six years or older. The aim is to reduce bacterial load in the lungs so as to reduce inflammation and deterioration of lung function. This is an update of a previously published review.To evaluate the effects of long-term inhaled antibiotic therapy in people with cystic fibrosis on clinical outcomes (lung function, frequency of exacerbations and nutrition), quality of life and adverse events (including drug-sensitivity reactions and survival).We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched ongoing trials registries. Date of last search: 28 June 2022.We selected trials where people with cystic fibrosis received inhaled anti-pseudomonal antibiotic treatment for at least three months, treatment allocation was randomised or quasi-randomised, and there was a control group (either placebo, no placebo or another inhaled antibiotic).Two authors independently selected trials, judged the risk of bias, extracted data from these trials and judged the certainty of the evidence using the GRADE system.The searches identified 410 citations to 125 trials; 18 trials (3042 participants aged between five and 45 years) met the inclusion criteria. Limited data were available for meta-analyses due to the variability of trial design and reporting of results. A total of 11 trials (1130 participants) compared an inhaled antibiotic to placebo or usual treatment for a duration between three and 33 months. Five trials (1255 participants) compared different antibiotics, two trials (585 participants) compared different regimens of tobramycin and one trial (90 participants) compared intermittent tobramycin with continuous tobramycin alternating with aztreonam. One trial (18 participants) compared an antibiotic to placebo and also to a different antibiotic and so fell into both groups. The most commonly studied antibiotic was tobramycin which was studied in 12 trials. Inhaled antibiotics compared to placebo We found that inhaled antibiotics may improve lung function measured in a variety of ways (4 trials, 814 participants). Compared to placebo, inhaled antibiotics may also reduce the frequency of exacerbations (risk ratio (RR) 0.66, 95% confidence interval (CI) 0.47 to 0.93; 3 trials, 946 participants; low-certainty evidence). Inhaled antibiotics may lead to fewer days off school or work (quality of life measure) (mean difference (MD) -5.30 days, 95% CI -8.59 to -2.01; 1 trial, 245 participants; low-certainty evidence). There were insufficient data for us to be able to report an effect on nutritional outcomes and there was no effect on survival. There was no effect on antibiotic resistance seen in the two trials that were included in meta-analyses. We are uncertain of the effect of the intervention on adverse events (very low-certainty evidence), but tinnitus and voice alteration were the only events occurring more often in the inhaled antibiotics group. The overall certainty of evidence was deemed to be low for most outcomes due to risk of bias within the trials and imprecision due to low event rates. Different antibiotics or regimens compared Of the eight trials comparing different inhaled antibiotics or different antibiotic regimens, there was only one trial for each unique comparison. We found no differences between groups for any outcomes except for the following. Aztreonam lysine for inhalation probably improved forced expiratory volume at one second (FEVsub1/sub) % predicted compared to tobramycin (MD -3.40%, 95% CI -6.63 to -0.17; 1 trial, 273 participants; moderate-certainty evidence). However, the method of defining the endpoint was different to the remaining trials and the participants were exposed to tobramycin for a long period making interpretation of the results problematic. We found no differences in any measure of lung function in the remaining comparisons. Trials measured pulmonary exacerbations in different ways and showed no differences between groups except for aztreonam lysine probably leading to fewer people needing treatment with additional antibiotics than with tobramycin (RR 0.66, 95% CI 0.51 to 0.86; 1 trial, 273 participants; moderate-certainty evidence); and there were fewer hospitalisations due to respiratory exacerbations with levofloxacin compared to tobramycin (RR 0.62, 95% CI 0.40 to 0.98; 1 trial, 282 participants; high-certainty evidence). Important treatment-related adverse events were not very common across comparisons, but were reported less often in the tobramycin group compared to both aztreonam lysine and colistimethate. We found the certainty of evidence for these comparisons to be directly related to the risk of bias within the individual trials and varied from low to high.Long-term treatment with inhaled anti-pseudomonal antibiotics probably improves lung function and reduces exacerbation rates, but pooled estimates of the level of benefit were very limited. The best evidence available is for inhaled tobramycin. More evidence from trials measuring similar outcomes in the same way is needed to determine a better measure of benefit. Longer-term trials are needed to look at the effect of inhaled antibiotics on quality of life, survival and nutritional outcomes.

Background: Gastrointestinal symptoms in cystic fibrosis (CF) are common and intrusive to daily life. Relieving gastrointestinal symptoms was identified as an important research priority and previously explored in an international survey in 2018. However, following the widespread introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators in 2019, the landscape of CF treatment has changed. We repeated an online survey to further describe gastrointestinal symptoms and their effect on quality of life (QoL) in the CFTR modulator era. Methods: An electronic survey consisting of closed questions and free text responses was distributed via social media and professional networks for a period of one month between March - April 2022. People with CF (pwCF), their family and friends, and healthcare professionals (HCPs) were invited to take part. Results: There were 164 respondents: 88 pwCF (54%), 22 (13%) family, and 54 (33%) healthcare professionals (HCPs). A total of 89/110 (81%) pwCF or family members reported CFTR modulator treatment. The most commonly reported symptoms were wind / gas, rumbling stomach noises, loose motions (modulator) and bloating (no modulator). Abdominal pain and bloating had the greatest impact on QoL. For those on a CFTR modulator, the proportion of pwCF reporting “no change” or “worse” for all of the symptoms surveyed was greater than the proportion reporting an improvement. Following modulator introduction, dietary changes were recommended by 28/35 (80%) of HCPs and reported by 38/76 (50%) lay respondents. Changes in medication were recommended by 19/35 (54%) HCPs and reported by 44/76 (58%) of patients and family members. Conclusion This survey has shown that gastrointestinal symptoms remain prevalent in pwCF in the CFTR modulator era, though the nature of these symptoms may have changed. A better understanding of the underlying pathophysiology of these symptoms is essential. Future clinical studies should focus on improving symptoms and QoL.

BACKGROUND: Respiratory disease is the main cause of morbidity and mortality in cystic fibrosis (CF), and many different therapies are used by people with CF in the management of respiratory problems. Bronchodilator therapy is used to relieve symptoms of shortness of breath and to open the airways to allow clearance of mucus. Despite the widespread use of inhaled bronchodilators in CF, there is little objective evidence of their efficacy. A Cochrane Review looking at both short‐ and long‐acting inhaled bronchodilators for CF was withdrawn from the Cochrane Library in 2016. That review has been replaced by two separate Cochrane Reviews: one on long‐acting inhaled bronchodilators for CF, and this review on short‐acting inhaled bronchodilators for CF. For this review 'inhaled' includes the use of pressurised metered dose inhalers (MDIs), with or without a spacer, dry powder devices and nebulisers. OBJECTIVES: To evaluate short‐acting inhaled bronchodilators in children and adults with CF in terms of clinical outcomes and safety. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books on 28 March 2022 and searched trial registries for any new or ongoing trials on 12 April 2022. We also searched the reference lists of relevant articles and reviews. SELECTION CRITERIA: We searched for randomised controlled trials (RCTs) or quasi‐RCTs that looked at the effect of any short‐acting inhaled bronchodilator delivered by any device, at any dose, at any frequency and for any duration compared to either placebo or another short‐acting inhaled bronchodilator in people with CF. We screened references as per standard Cochrane methodology. DATA COLLECTION AND ANALYSIS: Two review authors extracted data and assessed risk of bias using the Cochrane RoB 1 tool. Where we were not able to enter data into our analyses we reported results directly from the papers. We assessed the certainty of evidence using GRADE. MAIN RESULTS: We included 11 trials from our systematic search, with 191 participants meeting our inclusion criteria; three of these trials had three treatment arms. Eight trials compared short‐acting inhaled beta‐2 agonists to placebo and four trials compared short‐acting inhaled muscarinic antagonists to placebo. Three trials compared short‐acting inhaled beta‐2 agonists to short‐acting inhaled muscarinic antagonists. All were cross‐over trials with only small numbers of participants. We were only able to enter data into the analysis from three trials comparing short‐acting inhaled beta‐2 agonists to placebo. Short‐acting inhaled beta‐2 agonists versus placebo All eight trials (six single‐dose trials and two longer‐term trials) reporting on this comparison reported on forced expiratory volume in 1 second (FEV(1)), either as per cent predicted (% predicted) or L. We were able to combine the data from two trials in a meta‐analysis which showed a greater per cent change from baseline in FEV(1) L after beta‐2 agonists compared to placebo (mean difference (MD) 6.95%, 95% confidence interval (CI) 1.88 to 12.02; 2 trials, 82 participants). Only one of the longer‐term trials reported on exacerbations, as measured by hospitalisations and courses of antibiotics. Only the second longer‐term trial presented results for participant‐reported outcomes. Three trials narratively reported adverse events, and these were all mild. Three single‐dose trials and the two longer trials reported on forced vital capacity (FVC), and five trials reported on peak expiratory flow, i.e. forced expiratory flow between 25% and 75% (FEF(25-75)). One trial reported on airway clearance in terms of sputum weight. We judged the certainty of evidence for each of these outcomes to be very low, meaning we are very uncertain about the effect of short‐acting inhaled beta‐2 agonists on any of the outcomes we assessed. Short‐acting inhaled muscarinic antagonists versus placebo All four trials reporting on this comparison looked at the effects of ipratropium bromide, but in different doses and via different delivery methods. One trial reported FEV(1) % predicted; three trials measured this in L. Two trials reported adverse events, but these were few and mild. One trial reported FVC and three trials reported FEF(25-75). None of the trials reported on quality of life, exacerbations or airway clearance. We judged the certainty of evidence for each of these outcomes to be very low, meaning we are very uncertain about the effect of short‐acting inhaled muscarinic antagonists on any of the outcomes we assessed. Short‐acting inhaled beta‐2 agonists versus short‐acting inhaled muscarinic antagonists None of the three single‐dose trials reporting on this comparison provided data we could analyse. The original papers from three trials report that both treatments lead to an improvement in FEV(1) L. Only one trial reported on adverse events; but none were experienced by any participant. No trial reported on any of our other outcomes. We judged the certainty of evidence to be very low, meaning we are very uncertain about the effect of short‐acting inhaled beta‐2 agonists compared to short‐acting inhaled muscarinic antagonists on any of the outcomes we assessed. AUTHORS' CONCLUSIONS: All included trials in this review are small and of a cross‐over design. Most trials looked at very short‐term effects of inhaled bronchodilators, and therefore did not measure longer‐term outcomes. The certainty of evidence across all outcomes was very low, and therefore we have been unable to describe any effects with certainty.

Airway clearance has been an integral part of cystic fibrosis (CF) care for almost as long as CF has been identified as a condition. From diagnosis as a neonate through to end-of-life care, airway clearance is an everyday aspect of life, adding a considerable treatment burden to the lives of people with CF. There are many different techniques used for airway clearance which have evolved over time with an aim to improve effectiveness, support adherence and, more recently, to consider the impact of burden. A popular thought is whether airway clearance could be replaced by exercise. With new precision therapies in the form of CFTR modulators available, the CF landscape is rapidly changing, raising the question of whether certain treatments are needed at all. Depending on factors such as CFTR mutation, age, and pre-existing lung damage before starting a CFTR modulator, individuals with CF may need different levels of intensity and type of maintenance treatment. Precision medicine is likely to lead to the need for increased precision and individualized management around other maintenance therapies such as airway clearance.

In a recent James Lind Alliance Priority Setting Partnership in cystic fibrosis (CF) the top priority clinical research question was: "What are effective ways of simplifying the treatment burden of people with CF?" We aimed to summarise the lived experience of treatment burden and suggest research themes aimed at reducing it. An online questionnaire was co-produced and responses subjected to quantitative and thematic analysis. 941 survey responses were received (641 from lay community). People with CF reported a median of 10 (interquartile range: 6-15) current treatments. Seven main themes relating to simplifying treatment burden were identified. Treatment burden is high, extending beyond time taken to perform routine daily treatments, with impact varying according to person-specific factors. Approaches to communication, support, evaluation of current treatments, service set-up, and treatment logistics (obtaining/administration) contribute to burden, offering scope for evaluation in clinical trials or service improvement.

Reducing treatment burden in cystic fibrosis (CF) is the top research priority for patients and clinicians. Difficulty accessing medication is one aspect of treatment burden. We investigated this with an online survey available globally for patients with CF and healthcare professionals. Almost three quarters of patients with CF in our survey report difficulty getting repeat prescriptions on time, and most community pharmacists experience interrupted supplies of CF-specific medications. These barriers affect emotional and physical health of people with CF. Two-thirds of people with CF would like to get all their CF medication from one place, their CF centre.

Introduction: Cystic fibrosis is a life-limiting genetic condition characterized by recurrent pulmonary infection. Acquisition of infection can occur from environmental reservoirs, person-to-person transmission and from the healthcare environment. Primary prevention of infections through infection prevention and control measures is an important strategy in cystic fibrosis care.Areas covered: Here we present a systematic review of the evidence base around infection prevention and control in cystic fibrosis. We found 36 studies and 7 guidelines that met our inclusion criteria. Strategies covered include cohort segregation, individual segregation, hand hygiene, facemasks, combination strategies, equipment strategies, and adherence. Quality of evidence overall was deemed low or very low. Most guideline recommendations have little or no evidence to support them.Expert opinion: Although low quality, there is an abundance of evidence suggesting segregation is beneficial in reducing pathogen spread. Undertaking high-quality studies may, therefore, be ethically challenging. Large-scale registry studies may provide a better strategy for answering questions on the efficacy of infection control policy. With the rise of antibiotic resistance, effective eradication of cystic fibrosis pathogens is becoming more difficult so primary prevention through infection control will become increasingly important over the coming years.

IntroductionCystic fibrosis (CF) is a multisystem disorder. Treatment is complex and evidence for treatment decisions may be absent. Characterising gaps in the research evidence will highlight treatment uncertainties and help prioritise research questions. We systematically identified the evidence gaps for treatment decisions in CF.MethodsWe searched for systematic reviews and guidelines on treatment interventions in CF. Two researchers identified eligible reviews with arbitration from a third. Using a structured framework, we extracted and characterised evidence gaps.ResultsThere were 73 reviews and 21 guidelines that met our inclusion criteria. From these, we identified 148 evidence gaps across a range of treatment areas. We found 111 evidence gaps through systematic reviews and a further 37 from guidelines. The reason for an evidence gap could only be reliably characterised for systematic reviews. In most cases, there was more than one explanation—most commonly few or no trials (97/111 evidence gaps). Other important factors leading to evidence gaps were small sample size (49/111), inadequate duration of follow-up (38/111) or intervention (37/111) and factors relating to outcomes (35/111). Evidence gaps from both systematic reviews and guidelines fell into the following categories: Respiratory (91); Gastrointestinal (20); PhysiotherapyandExercise (16); Musculoskeletal (6); Endocrine (4); Basic defect of CF (8); Psychosocial (2); Ears, Nose and Throat (1).ConclusionsWe have compiled an up-to-date list of treatment uncertainties in CF and the reasons for these uncertainties. These can be used as a resource to aid researchers and funders when planning future trials.PROSPERO registration numberPre-results; CRD42015030111.

Cystic Fibrosis (CF) is the commonest life limiting inherited condition in the UK, affecting 3895 children. Children with CF undertake burdensome, time-consuming treatment, much of which is not evidence-based. Questions for clinical research should reflect patient and clinician priorities because both the number of potential participants and funding are limited. Aims We describe our work over a 4 year period to engage the CF community in prioritisation and co-production of clinical research. Methods In 2016–17 we undertook a James Lind Alliance (JLA) Priority Setting Partnership (PSP) to find the top 10 priorities for clinical research in CF. Through a series of surveys and workshops these were agreed in conjunction with the global CF community. In 2018–2019 we undertook further global surveys to explore 4 of the top 10 questions in more depth (treatment burden, gastrointestinal symptoms, adherence and exercise in place of some airway clearance). To avoid cross-infection and increase global reach, we adapted JLA methodology to use video conferencing and social media. Results During the past four years we have gathered opinions from 3729 respondents across six continents. Our youngest respondents were six years old and the median age of respondents with CF (answering directly or by proxy) was 15 years. On completion we disseminated the top 10 questions widely and worked to raise awareness with research funders such as the NIHR. This has resulted in two NIHR themed calls, featuring many of the JLA CF top 10 questions. Conclusions Encouraging patient participation, including children, in all stages of evidence-based medicine is vital to ensure clinical research is of good quality and relevant to patient needs.

IntroductionRelieving gastrointestinal (GI) symptoms was identified as a ‘top ten’ priority by our James Lind Alliance Priority Setting Partnership in cystic fibrosis (CF). We conducted an online survey to find out more about the effect of GI symptoms in CF.MethodsWe co-produced an online survey distributed to the CF community via web-based platforms. The survey consisted of open and closed questions designed to help us learn more about the effects of GI symptoms for people with CF (pwCF). We analysed the data using descriptive statistics and thematic analysis. We promoted the survey via social media and web-based platforms which allowed respondents from any country to take part. Our participants came from the CF community, including: adults and children with CF, parents and close family of pwCF and healthcare professionals (HCPs) working with pwCF.ResultsThere were 276 respondents: 90 (33%) pwCF, 79 (29%) family, 107 (39%) HCPs. The most commonly reported symptoms by lay respondents were stomach cramps/pain, bloating and a ‘combination of symptoms’. The top three symptoms that HCPs said were reported to them were reduced appetite, bloating and constipation. Almost all (94% (85/90)) HCPs thought medications helped to relieve GI symptoms but only 58% (82/141) of lay respondents agreed.ConclusionsOur survey has shown that GI symptoms among our participants are prevalent and intrude on daily lives of pwCF. There is a need for well-designed clinical studies to provide better evidence for management of GI symptoms and complications.

Introduction“What effective ways of motivation, support and technologies help people with cystic fibrosis improve and sustain adherence to treatment?” was identified as one of the James Lind Alliance Priority Setting Partnership’s top 10 research questions in cystic fibrosis (CF). Using electronic questionnaires, we aimed to gain a deeper understanding of this research priority.MethodThe work was led by the steering group representative of the UK CF community consisting of patients, carers and healthcare professionals (HCPs). Electronic questionnaires were completed over a 4-week period and promoted via online forums such as Twitter, the UK CF Trust and US CF Foundation websites and via professional networks. Analysis of the closed questions was completed using Microsoft Excel, with keyword analysis and the final thematic analysis completed using NVivo software.ResultsThere were 313 respondents; 176/313 (56%) were from people with CF and their families. HCPs comprised of 10 professional groups accounting for 137/313 (44%) of respondents, with global involvement of participants with the majority from the UK. Common themes identified as impacting on adherence included: having no time, treatment burden, competing life demands, fatigue and the patient’s general health. Having a routine was identified as the most frequently used motivational strategy, valued by both the patient and professional community. However, some strategies were valued more by HCPs than used in practice by patients; these included the use of short-term goal setting and technology use.ConclusionAdherence to treatment is crucial, however it is often suboptimal and strategies valued by HCPs to promote adherence are not always shared by patients. To promote adherence clinicians and researchers should be mindful that in a condition where treatment burden and time pressures are considerable, any interventions should focus on simplifying care and reducing treatment burden.

Cystic fibrosis (CF) is the commonest life-limiting inherited disorder in the UK. It affects many parts of the body including the lungs and gut leading to increased infection and problems digesting food. People with CF need to undergo many treatments each day throughout their whole lives. These include tablets, inhalers and breathing exercises, which are a huge burden, taking up several hours every day It is therefore, really important that the treatments we give are supported by good evidence, usually gathered from clinical trials. Unfortunately, we do not have good evidence for many of the CF treatments. We recently ran an exercise known as a James Lind Alliance Priority Setting Partnership (JLA PSP) to find out which the CF community feel are the top priority research questions. People with CF and those who look after them suggested questions to be answered by clinical trials. Through a series of online surveys and workshops these were then shortlisted to give a final top ten. Due to infection risk people with CF are advised not to mix, this meant we had to do things differently to the usual way JLA PSPs are carried out. We used videoconferencing to enable multiple people with CF to participate. Surveys were accessible online and promoted through social media. Background The James Lind Alliance (JLA) method is well recognised for setting research priorities. The JLA approach involves a combination of surveys and workshop interactions between patients, carers and health care professionals to identify and agree on a “top ten” list of research questions. Respiratory infection is one of the hallmarks of cystic fibrosis (CF). To avoid cross infection, patients are advised not to meet face to face, preventing us following standard JLA methodology. Here we describe adaptations made during our recent JLA Priority Setting Partnership (PSP) in CF. Methods We elicited and prioritised research questions, using sequential online surveys, promoted through social media. People with CF participated in steering committee meetings and the final workshop, using videoconferencing. Alterations to workshop methodology enabled participants attending in person and those joining remotely, to contribute equally. We also altered the JLA methodology to include “lone” questions, asked by only one survey respondent. We are now working with the CF community to co-produce research projects that answer these top ten. Results There were 482 respondents, from 23 countries, who submitted 1080 questions. Increases in the number of responses occurred just after promotion on social media. Use of videoconferencing enabled participation of multiple people with CF and ensured participation from anywhere in the world, including hospital inpatients. Inclusion of lone questions resulted in one being included in our top ten. Conclusions There is no “one-size-fits-all” for patient involvement methodologies. Through altering the JLA methods to fit our patient group we achieved wide participation. We believe that methods used in our project may also be applied to future partnerships to increase participation, especially where people may be hospitalised or be unable to travel. The methodology we are developing through the JLA PSP CF2 project may be useful for other PSPs to follow.

Airway clearance techniques (ACTs) are recommended in cystic fibrosis (CF) to prevent accumulation of secretions and lung infection. "Can exercise replace chest physiotherapy for people with CF?" is one of the CF community's top 10 research questions. We conducted an online survey of the CF community to gather data on current ACT use, recommendations, reported adherence levels and exercise strategies used. There were 488 respondents: 194 (40%) people with CF (pwCF), 141 (29%) family and 153 (31%) healthcare professionals (HCPs) (mostly physiotherapists). Only 10/285 (4%) of pwCF do no exercise at present and 163/303 (54%) already incorporate exercise into ACTs. ACTs were omitted by 128/267 (48%) of pwCF when they exercised. Nearly all (110/129, 93%) of HCPs currently recommend exercise to support ACTs. A trial replacing some or all ACTs with exercise, was supported by 80/110 (73%) of HCPs, with an additional 9/110 (8%) willing to consider in selected patients.

As awareness of the risks of cross infection has increased, infection prevention and control measures have become more draconian. Infection control measures can have a profound effect of the organisation and delivery of CF services and on the lives of people with CF outside the hospital. However, the consequences of inadequate infection control measures may be the permanent acquisition of a chronic infection which is virtually untreatable. Recommendations for infection prevention and control therefore must protect patients but should also be evidence-based and proportionate. This article will review the literature, juxtaposing evidence and popular practise.

There are many uncertainties regarding Cystic Fibrosis (CF) treatment. Recently, the first James Lind Alliance (JLA) Priority Setting Partnership (PSP) in CF was completed, bringing clinicians, patients and carers together to identify the Top 10 research priorities. Here we investigate how well the current clinical trials landscape reflects these priorities. Trials in CF were identified through searches of research databases (Pubmed, ANZCTR, EU clinical trials register, ClinicalTrials.gov and ISRCTN). Trials meeting inclusion criteria of registered intervention studies in CF published between 01.012016 and 11.09.2017 were matched to the Top 10 priorities. We identified 259 trials, with 193 fulfilling the inclusion criteria. Only 63 (33%) of these matched one or more of the JLA priorities showing that current clinical trials poorly reflect the JLA Top 10. By increasing awareness of the Top 10 priorities, it is hoped that this will fuel future research in areas important to the CF community.

There remain many treatment uncertainties in cystic fibrosis (CF). With limited resources, research should focus on questions which are most important to the CF community. We conducted a James Lind Alliance Priority Setting Partnership in CF. Research questions were elicited and then prioritised in successive surveys. A workshop agreed the final top 10. Online methods avoided cross infection and widened participation. The elicitation survey had 482 respondents (1080 questions) and prioritisation survey 677 respondents. Participants were drawn equally from the patient and clinical communities globally. We have achieved a consensus on 10 research priorities which will be attractive to funders.

The diagnosis of myelodysplastic syndromes (MDS) remains problematic due to the subjective nature of morphologic assessment. The reported high frequency of somatic mutations and increased structural variants by array-based cytogenetics have provided potential objective markers of disease; however, this has been complicated by reports of similar abnormalities in the healthy population. We aimed to identify distinguishing features between those with early MDS and reported healthy individuals by characterizing 69 patients who, following a nondiagnostic marrow, developed progressive dysplasia or acute myeloid leukemia. Targeted sequencing and array-based cytogenetics identified a driver mutation and/or structural variant in 91% (63/69) of prediagnostic samples with the mutational spectrum mirroring that in the MDS population. When compared with the reported healthy population, the mutations detected had significantly greater median variant allele fraction (40% vs 9% to 10%), and occurred more commonly with additional mutations (≥2 mutations, 64% vs 8%). Furthermore, mutational analysis identified a high-risk group of patients with a shorter time to disease progression and poorer overall survival. The mutational features in our cohort are distinct from those seen in the healthy population and, even in the absence of definitive disease, can predict outcome. Early detection may allow consideration of intervention in poor-risk patients.

The Hedgehog (Hh) signaling pathway influences multiple stages of murine T-cell development. Hh signaling mediates transcriptional changes by the activity of the Gli family of transcription factors, Gli1, Gli2 and Gli3. Both Gli2 and Gli3 are essential for mouse development and can be processed to function as transcriptional repressors or transcriptional activators, whereas Gli1, itself a transcriptional target of Hh pathway activation, can only function as a transcriptional activator and is not essential for mouse development. Gli1-deficient mice are healthy and appear normal and nonredundant functions for Gli1 have been difficult to identify. Here we show that Gli1 is non-redundant in the regulation of T-cell development in the thymus, at multiple developmental stages. Analysis of Gli1-deficient embryonic mouse thymus shows a role for Gli1 to promote the differentiation of CD4⁻CD8⁻ double negative (DN) thymocytes before pre- TCR signal transduction, and a negative regulatory function after pre-TCR signaling. In addition, introduction of a Class I-restricted transgenic TCR into the adult Gli1-deficient and embryonic Gli2-deficient thymus showed that both Gli1 and Gli2 influence its selection to the CD8 lineage.

Hedgehog signaling regulates differentiation, survival, and proliferation of the earliest double-negative (DN) thymocytes, but its importance at later stages of T-cell development is controversial. Here we use loss- and gain-of-function mouse models to show that Shh, by signaling directly to the developing thymocyte, is a negative regulator of pre-TCR–induced differentiation from DN to double-positive (DP) cell. When hedgehog signaling was reduced, in the Shh−/− and Gli2−/− thymus, or by T lineage–specific transgenic expression of a transcriptional-repressor form of Gli2 (Gli2ΔC2), differentiation to DP cell after pre-TCR signal transduction was increased. In contrast, when Hh signaling was constitutively activated in thymocytes, by transgenic expression of a constitutive transcriptional-activator form of Gli2 (Gli2ΔN2), the production of DP cells was decreased. Gene expression profiling showed that physiologic Hh signaling in thymocytes maintains expression of the transcription factor FoxA2 on pre-TCR signal transduction.

Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2DeltaC2), increased T-cell differentiation and activation in response to TCR signalling. Expression of the Gli2DeltaC2 transgene increased differentiation from CD4(+)CD8(+) to single positive thymocyte, and increased peripheral T cell populations. Gli2DeltaC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate.

The Hedgehog (Hh) signaling pathway is a key regulator of both embryonic development and homeostasis of adult tissues, including thymus and blood. In the thymus, Hh signals for differentiation, survival and proliferation in the early stages of T cell development, before TCR gene rearrangement. Our recent data has shown that Hh signaling also modulates T cell receptor (TCR) signal strength in more mature T lineage cells. We showed that constitutive activation of the Hh pathway in thymocytes (by transgenic expression of the transcriptional activator form of Gli2) decreased TCR signal strength with profound consequences for the thymus--allowing self-reactive T cells to escape deletion and altering T cell CD4/CD8 lineage decisions. In contrast, in the Sonic Hh deficient thymus, TCR signaling was increased, again influencing both TCR repertoire selection and CD4/8 lineage commitment. In peripheral T cells, the transcriptional changes induced by activation of the Hh signaling pathway lead to reduced T cell activation. Hh signaling also attenuated ERK phosphorylation and proliferation in mature T cells on TCR ligation. Modulation of TCR signal strength by Hh pathway activation has importance for immunity as the presence or absence of Hh in the environment in which a T cell is activated would shape the immune response.

Expression of TCRbeta and pre-TCR signalling are essential for differentiation of CD4- CD8- double negative (DN) thymocytes to the CD4+ CD8+ double-positive (DP) stage. Thymocyte development in adult Rag1, Rag2 or TCRbetadelta-deficient mice is arrested at the DN3 stage leading to the assumption that pre-TCR signalling and beta-selection occur at, and are obligatory for, the transition from DN3 to DN4. We show that the majority of DN3 and DN4 cells that differentiate during early embryogenesis in wild-type mice do not express intracellular (ic) TCRbeta/gammadelta. These foetal icTCRbeta-/gammadelta- DN4 cells were T lineage as determined by expression of Thy1 and icCD3 and TCRbeta DJ rearrangement. In addition, in the foetal Rag1-/- thymus, a normal percentage of DN4 cells were present. In wild-type mice after hydrocortisone-induced synchronisation of differentiation, the majority of DN4 cells that first emerged did not express icTCRbeta/gammadelta, showing that adult thymocytes can also differentiate to the DN4 stage independently of pre-TCR signalling. Pre-TCR signalling induced expansion in the DN4 population, but lack of TCRbeta/gammadelta expression did not immediately induce apoptosis. Our data demonstrate in vivo differentiation from DN3 to DN4 cell in the absence of TCRbeta/gammadelta expression in the foetal thymus, and after hydrocortisone treatment of adult mice.

TCR signal strength is involved in many cell fate decisions in the T-cell lineage. Here, we show that transcriptional events induced by Hedgehog (Hh) signaling reduced TCR signal strength in mice. Activation of Hh signaling in thymocytes in vivo by expression of a transgenic transcriptional-activator form of Gli2 (Gli2\#916;N2) changed the outcome of TCR ligation at many stages of thymocyte development, allowing self-reactive cells to escape clonal deletion; reducing transgenic TCR-mediated positive selection; reducing the ratio of CD4/CD8 single-positive (SP) cells; and reducing cell surface CD5 expression. In contrast, in the Shh\#8722;/\#8722; thymus the ratio of CD4/CD8 cells and both positive and negative selection of a transgenic TCR were increased, demonstrating that Shh does indeed influence TCR repertoire selection and the transition from double-positive (DP) to SP cell in a physiological situation. In peripheral T cells, Gli2\#916;N2 expression attenuated T-cell activation and proliferation, by a mechanism upstream of ERK phosphorylation.

We have recently shown that endogenous Annexin-A1 (AnxA1) plays a homeostatic regulatory role in mature T cells by modulating the strength of TCR signaling. In this study we investigated the role of endogenous AnxA1 in thymocyte maturation. Analysis of AnxA1(-/-) thymocyte populations at the immature CD4(-)CD8(-) double negative (DN) stage showed a proportional decrease in the DN1 and an increase in the DN3 subsets compared to control littermates. There were no significant differences in thymocyte numbers or proportions of CD4(+) and CD8(+) single positive (SP) populations between Anx1(-/-) and AnxA1(+/+) mice. However, when we crossed AnxA1(-/-) mice onto HY-TCR transgenic mice, we observed an increase in CD4(+)CD8(+) double positive (DP) and CD4 SP cells in male AnxA1(-/-)/HY-TCR compared to AnxA1(+/+)/HY-TCR. Conversely, female AnxA1(-/-)/HY-TCR mice showed an increase in DP and a decrease in CD8 (SP) cells compared to female AnxA1(+/+)/HY-TCR. Biochemical analysis of the signaling pathways responsible for these effects showed a decrease in anti-CD3-induced Erk phosphorylation and NFkappaB activation in AnxA1(-/-) thymocytes compared to control littermates. Together these findings demonstrate a role for endogenous AnxA1 in regulating both positive and negative selection of the TCR repertoire. These results suggest that targeting AnxA1 expression or function in T cells could represent a useful approach for the development of novel therapies for the treatment of autoimmune diseases.

The Hedgehog (Hh) responsive transcription factor Gli3 is required for efficient thymocyte development in the fetus. In this study we show that Gli3, not detected in adult thymocytes, is expressed in the murine fetal and adult thymus stroma. PCR array analysis revealed Cxcl9, Rbp1, and Nos2 as novel target genes of Gli3. We show that Gli3 positively regulates the expression of these genes, most likely by suppressing an intermediate repressor. Deletion of autoreactive thymocytes depends on their interactions with the thymus stroma. Repression of the proapoptotic gene Nos2 in Gli3 mutants coincides with reduced apoptosis of double positive thymocytes undergoing negative selection in vitro and in vivo, and the production of autoreactive thymocytes. Taken together these data indicate that Gli3 controls thymocyte apoptosis and negative selection possibly via the regulation of Nos2. Defective Gli3 expression in the thymus stroma also resulted in decreased CD5 expression on mature thymocytes and inappropriate production of MHC class I-selected CD4+ cells, both consistent with reduced TCR signal strength. Overall our data indicate that Gli3 expressed in the thymus stroma regulates negative selection and TCR signal strength via Hh-dependent and -independent mechanisms, with implications for autoimmunity.

Background Identifying myelodysplasia (MDS) by morphological assessment, particularly in patients with Methods A retrospective search identified 82 patients with paired non-diagnostic and diagnostic samples of whom 69 had adequate molecular material at both time points for assessment. We developed a targeted gene panel which is both time efficient and economically viable as a diagnostic tool incorporating 26 of the most commonly mutated genes in myeloid malignancies. Primers were designed using the Access ArrayTMTarget-Specific Primers design service (Fluidigm). DNA either stored at the point of referral or extracted from archived slides was subjected to SNP array analysis (Illumina CytoSNP-12) and targeted deep sequencing (Illumina MiSeq). Initial read alignment and variant calling were performed using MiSeq reporter (Illumina) and variants were annotated using the Ensembl variant effect predictor. Following exclusion of known polymorphisms and correlation with the COSMIC database, mutations were confirmed using Sanger sequencing. CytoSNP data was visualised using KaryoStudio software (Illumina). Results A driver mutation, with or without a structural variant, was identified in 91% (63/69) of non-diagnostic samples and this increased to 94% (65/69) at the point of diagnosis. Crucially the majority of mutations identified in the non-diagnostic sample were likely oncogenic being either truncating variants (nonsense mutations, indels) or a mutation previously reported in the literature. There was a predominance of mutations involving epigenetic regulators and spliceosome genes at the earlier time point with mutations in TET2, SRSF2 and ASXL1 being detected in 38%, 26% and 20% of patients respectively. The spectrum of mutations identified in the non-diagnostic sample mirrored that reported in large cohorts of MDS patients with the exception of SF3B1 which was identified in only 3 patients (4%). This likely reflects the relative ease in identifying ring sideroblasts morphologically. The median time between samples was 476 days (range 19-2484). A total of 26 patients (38%) acquired mutations between samples. This occurred most commonly in those patients who developed AML (15/21 patients; 71%) with a resultant mutation rate of 100% in this disease subgroup. Transcription factor and cell signalling genes were the most commonly acquired mutations between samples (26/35 mutations; 74%). Importantly, with the exception of 1 patient, all those who progressed to AML had an abnormality detected on the non-diagnostic sample. Structural variants (copy number variation or loss of heterozygosity) were identified in 23% (16/69) of non-diagnostic samples increasing to 48% (33/69) at the point of diagnosis. However, these consistently occurred in the presence of a driver mutation. Conclusion This study has shown that MDS patients with ambiguous morphology at the time of presentation possess very frequent driver mutations that can be readily demonstrated using a limited, cost-effective sequencing panel. Mutational analysis may represent an objective and more reliable method of identifying patients with early stage disease. The paucity of structural variants along with the frequent demonstration of epigenetic regulators and spliceosome mutations in the non-diagnostic samples provides further insight into disease pathogenesis. Disclosures Jack: Roche: Research Funding; Genentech: Collaboration, Collaboration Other.