Deep Learning-based Segmentation of Computed Tomography Scans Predicts Disease Progression and Mortality in Idiopathic Pulmonary Fibrosis.

Rationale: Despite evidence demonstrating a prognostic role for computed tomography (CT) scans in idiopathic pulmonary fibrosis (IPF), image-based biomarkers are not routinely used in clinical practice or trials. Objectives: To develop automated imaging biomarkers using deep learning-based segmentation of CT scans. Methods: We developed segmentation processes for four anatomical biomarkers, which were applied to a unique cohort of treatment-naive patients with IPF enrolled in the PROFILE (Prospective Observation of Fibrosis in the Lung Clinical Endpoints) study and tested against a further United Kingdom cohort. The relationships among CT biomarkers, lung function, disease progression, and mortality were assessed. Measurements and Main Results: Data from 446 PROFILE patients were analyzed. Median follow-up duration was 39.1 months (interquartile range, 18.1-66.4 mo), with a cumulative incidence of death of 277 (62.1%) over 5 years. Segmentation was successful on 97.8% of all scans, across multiple imaging vendors, at slice thicknesses of 0.5-5 mm. Of four segmentations, lung volume showed the strongest correlation with FVC (r = 0.82; P < 0.001). Lung, vascular, and fibrosis volumes were consistently associated across cohorts with differential 5-year survival, which persisted after adjustment for baseline gender, age, and physiology score. Lower lung volume (hazard ratio [HR], 0.98 [95% confidence interval (CI), 0.96-0.99]; P = 0.001), increased vascular volume (HR, 1.30 [95% CI, 1.12-1.51]; P = 0.001), and increased fibrosis volume (HR, 1.17 [95% CI, 1.12-1.22]; P < 0.001) were associated with reduced 2-year progression-free survival in the pooled PROFILE cohort. Longitudinally, decreasing lung volume (HR, 3.41 [95% CI, 1.36-8.54]; P = 0.009) and increasing fibrosis volume (HR, 2.23 [95% CI, 1.22-4.08]; P = 0.009) were associated with differential survival. Conclusions: Automated models can rapidly segment IPF CT scans, providing prognostic near and long-term information, which could be used in routine clinical practice or as key trial endpoints.

Lymphangioleiomyomatosis in patients with tuberous sclerosis: a national centre audit.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is common in tuberous sclerosis complex (TSC) yet under recognised with management mostly based upon evidence obtained from patients with sporadic LAM. We performed a prospective audit of patients with TSC-LAM attending a national referral centre to inform management guidelines. METHODS: The UK LAM Centre was established in 2011 and conducts a prospective audit of pre-defined quality outcomes for all subjects. Audit data are reported on all patients with TSC-LAM and a comparator population of patients with sporadic LAM. RESULTS: Between 2011 and 2022, 73 patients were seen with TSC-LAM. All were women with a mean (SD) age of 39 (12) years. Referral rates were similar over the study period including after the introduction of CT screening. Median age of diagnosis with TSC was 11 years (range 0-70) with one third diagnosed with TSC as adults. Compared with all TSC patients in the 'TOSCA' registry, TSC-LAM patients tended to have been diagnosed with TSC at an older age, had fewer neuro-cognitive manifestations and were more likely to have angiomyolipoma. The most common presentations of TSC-LAM were following workup for angiomyolipoma, pneumothorax or dyspnoea with only one fifth detected after CT screening. Baseline FEV1 and DLCO at first assessment were reduced to 77 and 63% predicted respectively and were similar to patients with sporadic LAM. During follow-up, FEV1 fell by a mean of 81 ml/year and DLCO fell by 0.309 mmol/ml/kPa/year in patients not being treated with an mTOR inhibitor. 55% required treatment with either sirolimus or Everolimus for LAM or angiomyolipoma respectively. For those treated with an mTOR inhibitor, mean FEV1 fell by 3 ml/year and DLCO increased by 0.032 mmol/ml/kPa/year and was similar to sporadic LAM. Risk of death due to LAM or need for lung transplant in patients with TSC-LAM was 0.67%/year. CONCLUSIONS: Despite screening recommendations, LAM is often diagnosed in TSC after symptoms develop which may delay treatment. Complications including pneumothorax and loss of lung function are significant and similar to sporadic LAM. Work is needed to implement the recommended CT screening for LAM and improve respiratory care for TSC-LAM.

Diagnosis of cystic lung diseases: a position statement from the UK Cystic Lung Disease Rare Disease Collaborative Network.

BACKGROUND: Rare cystic lung diseases are increasingly recognised due the wider application of CT scanning making cystic lung disease management a growing part of respiratory care. Cystic lung diseases tend to have extrapulmonary features that can both be diagnostic but also require surveillance and treatment in their own right. As some of these diseases now have specific treatments, making a precise diagnosis is crucial. While Langerhans cell histiocytosis, Birt-Hogg-Dubé syndrome, lymphoid interstitial pneumonia and lymphangioleiomyomatosis are becoming relatively well-known diseases to respiratory physicians, a targeted and thorough workup improves diagnostic accuracy and may suggest other ultrarare diseases such as light chain deposition disease, cystic pulmonary amyloidosis, low-grade metastatic neoplasms or infections. In many cases, diagnostic information is overlooked leaving uncertainty over the disease course and treatments. AIMS: This position statement from the Rare Disease Collaborative Network for cystic lung diseases will review how clinical, radiological and physiological features can be used to differentiate between these diseases. NARRATIVE: We highlight that in many cases a multidisciplinary diagnosis can be made without the need for lung biopsy and discuss where tissue sampling is necessary when non-invasive methods leave diagnostic doubt. We suggest an initial workup focusing on points in the history which identify key disease features, underlying systemic and familial diseases and a clinical examination to search for connective tissue disease and features of genetic causes of lung cysts. All patients should have a CT of the thorax and abdomen to characterise the pattern and burden of lung cysts and extrapulmonary features and also spirometry, gas transfer and a 6 min walk test. Discussion with a rare cystic lung disease centre is suggested before a surgical biopsy is undertaken. CONCLUSIONS: We suggest that this focused workup should be performed in all people with multiple lung cysts and would streamline referral pathways, help guide early treatment, management decisions, improve patient experience and reduce overall care costs. It could also potentially catalyse a national research database to describe these less well-understood and unidentified diseases, categorise disease phenotypes and outcomes, potentially leading to better prognostic data and generating a stronger platform to understand specific disease biology.

The Burden and Impact of Cough in Patients with Idiopathic Pulmonary Fibrosis: An Analysis of the Prospective Observational PROFILE Study.

Rationale: Cough is a commonly reported symptom in idiopathic pulmonary fibrosis (IPF) that negatively impacts patient-reported quality of life (QoL). However, both the burden of cough at diagnosis and the behavior of cough over time have not been systematically described in patients with IPF. Objectives: By utilizing data prospectively collected as part of the PROFILE study, we sought to assess cough burden and the impact that this has on QoL within a cohort of patients with newly diagnosed IPF. We also reexamined the previously described relationship between cough and mortality and the association of cough with the MUC5B promoter polymorphism. Methods: The PROFILE study is a multicenter, prospective, observational, longitudinal cohort study of incident IPF. Scores on the Leicester Cough Questionnaire (LCQ) were recorded at baseline in 632 subjects and then repeated 6 monthly in a subset (n = 216) of the cohort. Results: The median LCQ score at diagnosis was 16.1 (interquartile range, 6.5). LCQ scores remained stable over the subsequent year in the majority of patients. There was a weak association between LCQ score and baseline lung function, with worse cough-related QoL associated with more severe physiological impairment. Cough scores were not associated with subsequent mortality after correcting for baseline lung function. Furthermore, there was no relationship between LCQ score and MUC5B promoter polymorphism status. Conclusions: The burden of cough in IPF is high. Although cough is weakly associated with disease severity at baseline, cough-specific QoL, as measured by the LCQ, confers no prognostic value. Cough-specific QoL burden remains relatively stable over time and does not associate with MUC5B promoter polymorphism.

Predicting Individualized Lung Disease Progression in Treatment-Naive Patients With Lymphangioleiomyomatosis.

BACKGROUND: Lung function decline varies significantly in patients with lymphangioleiomyomatosis (LAM), impeding individualized clinical decision-making. RESEARCH QUESTION: Can we aid individualized decision-making in LAM by developing a dynamic prediction model that can estimate the probability of clinically relevant FEV1 decline in patients with LAM before treatment initiation? STUDY DESIGN AND METHODS: Patients observed in the US National Heart, Lung, and Blood Institute (NHLBI) Lymphangioleiomyomatosis Registry were included. Using routinely available variables such as age at diagnosis, menopausal status, and baseline lung function (FEV1 and diffusing capacity of the lungs for carbon monoxide [Dlco]), we used novel stochastic modeling and evaluated predictive probabilities for clinically relevant drops in FEV1. We formed predictive probabilities of transplant-free survival by jointly modeling longitudinal FEV1 and lung transplantation or death events. External validation used the UK Lymphangioleiomyomatosis Natural History cohort. RESULTS: Analysis of the NHLBI Lymphangioleiomyomatosis Registry and UK Lymphangioleiomyomatosis Natural History cohorts consisted of 216 and 185 individuals, respectively. We derived a joint model that accurately estimated the risk of future lung function decline and 5-year probabilities of transplant-free survival in patients with LAM not taking sirolimus (area under the receiver operating characteristic curve [AUC], approximately 0.80). The prediction model provided estimates of forecasted FEV1, rate of FEV1 decline, and probabilities for risk of prolonged drops in FEV1 for untreated patients with LAM with a high degree of accuracy (AUC > 0.80) for the derivation cohort as well as the validation cohort. Our tool is freely accessible at: https://anushkapalipana.shinyapps.io/testapp_v2/. INTERPRETATION: Longitudinal modeling of routine clinical data can allow individualized LAM prognostication and assist in decision-making regarding the timing of treatment initiation.

Disease monitoring using lung function trajectory in lymphangioleiomyomatosis: assessment in two national cohorts.

STUDY QUESTION: In lymphangioleiomyomatosis, airflow obstruction and impairment of gas transfer progress at variable rates and serial lung function is recommended for disease monitoring. As these measurements are variable, recognising subjects needing treatment can be difficult. We used two prospective national cohorts to study change over time and variation in FEV1 to inform clinical decision making. PATIENTS AND METHODS: Clinical and lung function data for 141 UK and 148 American subjects were studied. Multilevel mixed effects modelling, route mean square analysis of errors and Bland-Altman analysis were used to analyse variability in lung function over time. RESULTS: At baseline assessment, DLCO was reduced to a greater degree than FEV1. In untreated patients, FEV1 and DLCO declined at proportionately similar rates independent of initial lung function. In mechanistic target of rapamycin (mTOR) inhibitor treated patients, FEV1 stabilised but DLCO continued to decline. FEV1/DLCO per cent predicted ratio was 1.37 (0.43) at baseline and increased to 1.41 (0.50) after 42 (24) months (p=0.0002). At least five measurements were required before >70% of individuals had estimates of rate of FEV1 loss within 50 mL/year and DLCO loss within 0.1 mmol/min/kPa/year of the final values. CONCLUSIONS: While FEV1 and DLCO fall proportionately in most, in early disease and during mTOR inhibitor treatment, DLCO should also be monitored as it may fall independent of FEV1. Since at least five observations over many months are required to make confident estimates of FEV1 and DLCO trajectories, new strategies are needed to measure disease activity and target early treatment appropriately.

A lung targeted miR-29 mimic as a therapy for pulmonary fibrosis.

BACKGROUND: MicroRNAs are non-coding RNAs that negatively regulate gene networks. Previously, we reported that systemically delivered miR-29 mimic MRG-201 reduced fibrosis in animal models, supporting the consideration of miR-29-based therapies for idiopathic pulmonary fibrosis (IPF). METHODS: We generated MRG-229, a next-generation miR-29 mimic based on MRG-201 with improved chemical stability due to additional sugar modifications and conjugation with the internalization moiety BiPPB (PDGFbetaR-specific bicyclic peptide)1. We investigated the anti-fibrotic efficacy of MRG-229 on TGF-ß1 treated human lung fibroblasts (NHLFs), human precision cut lung slices (hPCLS), and in vivo bleomycin studies; toxicology was assessed in two animal models, rats, and non-human primates. Finally, we examined miR-29b levels in a cohort of 46 and 213 patients with IPF diagnosis recruited from Yale and Nottingham Universities (Profile Cohort), respectively. FINDINGS: The peptide-conjugated MRG-229 mimic decreased expression of pro-fibrotic genes and reduced collagen production in each model. In bleomycin-treated mice, the peptide-conjugated MRG-229 mimic downregulated profibrotic gene programs at doses more than ten-fold lower than the original compound. In rats and non-human primates, the peptide-conjugated MRG-229 mimic was well tolerated at clinically relevant doses with no adverse findings observed. In human peripheral blood from IPF patients decreased miR-29 concentrations were associated with increased mortality in two cohorts potentially identified as a target population for treatment. INTERPRETATION: Collectively, our results provide support for the development of the peptide-conjugated MRG-229 mimic as a potential therapy in humans with IPF. FUNDING: This work was supported by NIH NHLBI grants UH3HL123886, R01HL127349, R01HL141852, U01HL145567.

A survey of use of mTOR inhibitors in patients with lymphangioleiomyomatosis listed for lung transplant.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is an uncommon indication for lung transplantation. The use of mechanistic target of rapamycin (mTOR) inhibitors, which are the mainstay of treatment in progressive LAM, in patients awaiting lung transplant is controversial. We sought to examine worldwide practice patterns in use of mTOR inhibitors in LAM patients on the lung transplant waiting list. METHODS: We designed and disseminated an online survey about institution-specific practice patterns, particularly regarding listing LAM patients for lung transplant and use of mTOR inhibitors in those patients on the transplant waitlist. RESULTS: Of the 49 unique respondent programs, 83.6% had previously listed a LAM patient for lung transplant. Thirteen centers allowed patients to continue on mTOR inhibitor until time of lung transplant. None of those centers reported any complications or deaths attributable to mTOR inhibitor adverse effects. CONCLUSION: There exists significant variability in practice patterns concerning the use of mTOR inhibitors in LAM patients on the lung transplant waiting list. Our survey suggests favorable outcomes for those patients that did continue mTOR inhibitor up to time of transplant. Further data regarding the risk of anastomotic complication with use of mTOR inhibitors in the pre-transplant period would help provide clarity in this debate.

Three-Month FVC Change: A Trial Endpoint for Idiopathic Pulmonary Fibrosis Based on Individual Participant Data Meta-analysis.

Rationale: Novel therapies for idiopathic pulmonary fibrosis (IPF) are in development, but there remains uncertainty about the optimal trial endpoint. An earlier endpoint would enable assessment of a greater number of therapies in adaptive trial designs. Objectives: To determine whether short-term changes in FVC, DlCO, and six-minute-walk distance could act as surrogate endpoints to accelerate early-phase trials in IPF. Methods: Individual participant data (IPD) from IPF clinical trials were included in a two-step random-effects meta-analysis to determine whether baseline or 3-month changes in FVC, DlCO, and 6-minute-walk distance were associated with mortality or disease progression in placebo arms. Three-month and 12-month FVC decline endpoints were compared with treatment arm data from antifibrotic studies by meta-regression. Measurements and Main Results: IPD were available from 12 placebo cohorts totaling 1,819 participants, with baseline and 3-month changes in all physiological variables independently associated with poorer outcomes. Treatment data were available from six cohorts with 1,684 participants. For each 2.5% relative decline in FVC over 3 months, there was an associated 15% (adjusted hazard ratio, 1.15; 95% confidence interval [CI], 1.06-1.24; I2 = 59.4%) and 20% (adjusted hazard ratio, 1.20; 95% CI, 1.12-1.28; I2 = 18.0%) increased risk for mortality in untreated and treated individuals, respectively. An FVC change treatment effect was observed between treatment and placebo arms at 3 months (difference in FVC change of 42.9 ml; 95% CI, 24.0-61.8 ml; P < 0.001). Conclusions: IPD meta-analysis demonstrated that 3-month changes in physiological variables, particularly FVC, were associated with mortality among individuals with IPF. FVC change over 3 months may hold potential as a surrogate endpoint in IPF adaptive trials.

Cross talk between LAM cells and fibroblasts may influence alveolar epithelial cell behavior in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a female-specific cystic lung disease in which tuberous sclerosis complex 2 (TSC2)-deficient LAM cells, LAM-associated fibroblasts (LAFs), and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial (AT2) cells. We hypothesized that the behavior of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in the patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared with parenchymal AT2 cells, demonstrated by increased Ki67 expression. Furthermore, nodular AT2 cells express proteins associated with epithelial activation in other disease states including matrix metalloproteinase 7, and fibroblast growth factor 7 (FGF7). In vitro, LAF-conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair, and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, a potential mediator of fibroblast-epithelial cross talk, in a mechanistic target of rapamycin (mTOR)-dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behavior. Fibroblast-derived FGF7 may contribute to the cross talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

Lymphangioleiomyomatosis: pathogenesis, clinical features, diagnosis, and management.

Lymphangioleiomyomatosis (LAM) is a slowly progressive, low-grade, metastasising neoplasm of women, characterised by infiltration of the lung parenchyma with abnormal smooth muscle-like cells, resulting in cystic lung destruction. The invading cell in LAM arises from an unknown source and harbours mutations in tuberous sclerosis complex (TSC) genes that result in constitutive activation of the mechanistic target of rapamycin (mTOR) pathway, dysregulated cellular proliferation, and a programme of frustrated lymphangiogenesis, culminating in disordered lung remodelling and respiratory failure. Over the past two decades, all facets of LAM basic and clinical science have seen important advances, including improved understanding of molecular mechanisms, novel diagnostic and prognostic biomarkers, effective treatment strategies, and comprehensive clinical practice guidelines. Further research is needed to better understand the natural history of LAM; develop more powerful diagnostic, prognostic, and predictive biomarkers; optimise the use of inhibitors of mTOR complex 1 in the treatment of LAM; and explore novel approaches to the development of remission-inducing therapies.

Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

Mast-Cell Tryptase Release Contributes to Disease Progression in Lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

Machine learning can predict disease manifestations and outcomes in lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare multisystem disease with variable clinical manifestations and differing rates of progression that make management decisions and giving prognostic advice difficult. We used machine learning to identify clusters of associated features which could be used to stratify patients and predict outcomes in individuals. PATIENTS AND METHODS: Using unsupervised machine learning we generated patient clusters using data from 173 women with LAM from the UK and 186 replication subjects from the US National Heart, Lung, and Blood Institute (NHLBI) LAM registry. Prospective outcomes were associated with cluster results. RESULTS: Two- and three-cluster models were developed. A three-cluster model separated a large group of subjects presenting with dyspnoea or pneumothorax from a second cluster with a high prevalence of angiomyolipoma symptoms (p=0.0001) and tuberous sclerosis complex (TSC) (p=0.041). Patients in the third cluster were older, never presented with dyspnoea or pneumothorax (p=0.0001) and had better lung function. Similar clusters were reproduced in the NHLBI cohort. Assigning patients to clusters predicted prospective outcomes: in a two-cluster model the future risk of pneumothorax was 3.3 (95% CI 1.7-5.6)-fold greater in cluster 1 than cluster 2 (p=0.0002). Using the three-cluster model, the need for intervention for angiomyolipoma was lower in clusters 2 and 3 than cluster 1 (p<0.00001). In the NHLBI cohort, the incidence of death or lung transplant was much lower in clusters 2 and 3 (p=0.0045). CONCLUSIONS: Machine learning has identified clinically relevant clusters associated with complications and outcome. Assigning individuals to clusters could improve decision making and prognostic information for patients.

Pregnancy in lymphangioleiomyomatosis: clinical and lung function outcomes in two national cohorts.

Pregnancy in women with lymphangioleiomyomatosis (LAM) has been associated with increased complications and worsening lung function although objective data to advise patients are not available. We assessed lung function and CT scans before and after pregnancy in 16 women with LAM. During the pregnancy, pneumothorax was frequent and mean forced expiratory volume in 1 s (FEV1) fell from 77%±19% prepregnancy to 64%±25% predicted and DLCO from 66±26 to 57±26 (both p<0.01). After pregnancy, rates of FEV1 decline were high and 10 patients required sirolimus. Women with LAM, especially with moderate or advanced disease should be counselled regarding adverse events and loss of lung function during the pregnancy.

Evolution of lung pathology in lymphangioleiomyomatosis: associations with disease course and treatment response.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease with a variable clinical course. The lungs are infiltrated by nodules of LAM cells, stromal cells and inflammatory cells, causing lung cysts and respiratory failure. We used immunohistochemical markers in lung biopsy and transplant samples from a national cohort of women with LAM with linked clinical data to understand how LAM nodule cell populations changed with disease progression. Marker distribution was examined qualitatively by dual immunohistochemistry, and markers for LAM cells, fibroblasts, lymphatics, mast cells, proliferation, cathepsin K and mTOR pathway activity were quantitated in LAM nodules and compared with clinical features and prospective lung function loss. The LAM cell marker PNL2 was more extensively expressed in those with higher forced expiratory volume in one second (FEV1 ), higher diffusion in the lung for carbon monoxide (DLCO ) and less extensive disease involvement whilst the converse was true for the protease cathepsin K. Each percentage increase in cathepsin K reactivity was associated with a 0.65% decrease in FEV1 (95% CI -1.11 to -0.18) and a 0.50% decrease in DLCO (95% CI -0.96 to -0.05). Higher reactivity to the mTOR complex 1 activation marker, phospho-ribosomal protein S6, was associated with a better lung function response to rapamycin (p = 0.0001). We conclude that LAM nodules evolve with disease progression, with LAM cells becoming outnumbered by fibroblasts. Increasing cathepsin K expression is associated with more severe disease and lung function loss. Markers of mTOR activation predict the response to rapamycin, suggesting that more advanced LAM may be less mTOR responsive and treatments specifically targeted towards LAM associated fibroblasts may have roles as adjuncts to mTOR inhibition.

Sphingolipid, fatty acid and phospholipid metabolites are associated with disease severity and mTOR inhibition in lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a rare multisystem disease almost exclusively affecting women which causes loss of lung function, lymphatic abnormalities and angiomyolipomas. LAM occurs sporadically and in people with tuberous sclerosis complex (TSC). Loss of TSC gene function leads to dysregulated mechanistic target of rapamycin (mTOR) signalling. As mTOR is a regulator of lipid and nucleotide synthesis, we hypothesised that the serum metabolome would be altered in LAM and related to disease severity and activity. METHODS: Ultrahigh performance liquid chromatography-tandem mass spectroscopy was used to examine the serum metabolome of 79 closely phenotyped women with LAM, including 29 receiving treatment with an mTOR inhibitor and 43 healthy control women. RESULTS: Sphingolipid, fatty acid and phospholipid metabolites were associated with FEV1 in women with LAM (eg, behenoyl sphingomyelin adjusted (adj.) p=8.10 × 10-3). Those with higher disease-burden scores had abnormalities in fatty acid, phospholipid and lysolipids. Rate of loss of FEV1 was associated with differences in acyl-carnitine, acyl-glycines, acyl-glutamine, fatty acids, endocanbinoids and sphingolipids (eg, myristoleoylcarnitine adj. p=0.07). In TSC-LAM, rapamycin affected modules of interrelated metabolites which comprised linoleic acid, the tricarboxylic acid cycle, aminoacyl-tRNA biosynthesis, cysteine, methionine, arginine and proline metabolism. Metabolomic pathway analysis within modules reiterated the importance of glycerophospholipid metabolites (adj. p=0.047). CONCLUSIONS: Women with LAM have altered lipid metabolism. The associations between these metabolites, multiple markers of disease activity and their potential biological roles in cell survival and signalling, suggest that lipid species may be both disease-relevant biomarkers and potential therapeutic targets for LAM.