BACKGROUND:Sex is an important part of life for many adults, yet sexual function may be impacted by chronic respiratory diseases such as pulmonary fibrosis (PF). This multinational study sought to characterise the impact of PF on sex and sexual function, using mixed quantitative and qualitative methodology. METHODS:We analysed data from a patient registry, a clinical study, an online survey and patient interviews to accomplish our objective and develop a conceptual framework for describing the effects of PF on sex-related quality of life. These cohorts used three distinct questionnaires, University of California, San Diego Shortness of Breath Questionnaire (UCSD-SOBQ), Sheffield Profile for Assessment and Referral to Care (SPARC) questionnaire, and Changes in Sexual Function Questionnaire (CSFQ), to assess sexual dysfunction, respectively. Individual patient interviews were conducted and qualitatively evaluated using thematic analysis. RESULTS:Dyspnoea with sexual activity affected 2054/2759 (74%) of registry patients, associated with male sex, lower forced vital capacity (FVC) % predicted, lower diffusing capacity of the lung for carbon monoxide (D LCO) % predicted and worse cough. Distress due to the effect of PF on their sex life was reported in 52/225 (23%) of the clinical cohort, associated with younger age, male sex, lower D LCO % predicted and worse cough. Sexual dysfunction was common, affecting 56/67 (83%) female and 63/73 (86%) male survey respondents. Qualitative analysis of patient interviews identified several themes, including sex life limitations, changes in interpersonal relationships and quality of life. All patients wanted to discuss sex with trusted healthcare providers. CONCLUSIONS:In this multinational study, patients with PF reported engaging in sex and sexual activities but were adversely impacted by the effect of PF on their sex life, with both physical and psychological limitations. Sexual dysfunction was common, driven by multiple disease domains. Sexual health appears to be an important component of comprehensive patient care.

BACKGROUND:Long term respiratory symptoms are reported following recovery of acute COVID-19 infection and residual lung abnormalities (RLA) on follow-up thoracic computed tomography (CT) after COVID-19 hospitalisation have been observed. It is unknown whether RLA are associated with epithelial lung injury. METHODS:Plasma was sampled from the observational Post HOSPitalisation-COVID cohort at five months post-hospitalisation. Epithelial injury biomarkers Krebs von den Lungen-6 (KL-6), matrix metalloproteinase 7 (MMP-7), surfactant protein-D (SP-D) and surfactant protein-A (SP-A) were assayed. In those without follow-up CT, RLA at-risk was defined by percent predicted DLCO <80% and/or abnormal chest X-ray, otherwise they were considered low-risk. Follow-up CT RLA was defined as combined involvement of ground glass opacity and reticulation ≥10%. FINDINGS:A total of 957 people were included, 846 people with no CT (at-risk n = 103; 12.2%), 111 people with follow-up CT (RLA ≥10% n = 85; 76.6%). All epithelial injury biomarkers were significantly elevated in people at-risk of RLA compared with low-risk. KL-6 and MMP-7 were significantly higher in people with ≥10% RLA than those with <10%, SP-D and SP-A did not reach significance. SP-D and SP-A were associated with percent involvement of reticulation (3.22%, 95% CI 1.19-5.24; 3.03%, 95% CI 0.76-5.30, respectively). INTERPRETATION:RLA after acute COVID-19 infection were consistent with elevated epithelial injury biomarkers and pro-fibrotic signalling. Future studies should address the temporal association between fibrotic biomarkers and resolution or progression of radiological involvement. FUNDING:MRC-UK Research and Innovation and National Institute for Health Research (NIHR) rapid response panel to tackle COVID-19 (MR/V027859/1; COV0319; MR/W006111/1).

Background:Idiopathic pulmonary fibrosis (IPF) is a rare, incurable lung disease with a median survival of 3-5 years after diagnosis. Treatment options are limited. Genetic association studies can identify new genes involved in disease that might represent potential new drug targets, and it has been shown that drug targets with support from genetic studies are more likely to be successful in clinical development. Previous genome-wide association studies (GWAS) of IPF susceptibility have identified more than 20 signals implicating genes involved in multiple mechanisms, including telomere dysfunction, cell-cell adhesion, host defence immunity, various signalling pathways and, more recently, mitotic spindle assembly complex. Aim:To leverage new datasets and genotype imputation to discover further genes involved in development of IPF that could yield new pathobiological avenues for exploration and to guide future drug target discovery. Methods:We conducted a GWAS of IPF susceptibility including seven IPF case-control studies comprising 5,159 IPF cases and 27,459 controls of European ancestry, where IPF diagnosis was made by a respiratory clinician according to international guidelines. Genotypes were obtained from Whole Genome Sequencing (WGS) or from array-based imputation to the TOPMed WGS reference panel. New signals were replicated in independent biobanks with IPF defined using Electronic Healthcare Records. Bayesian fine-mapping was performed to identify the most likely causal variant(s) and bioinformatic investigation undertaken to map associated variants to putative causal genes. Results:We identified three novel genetic signals of association with IPF susceptibility. Genes prioritised by functional evidence at these signals included MUC1, which encodes a large transmembrane glycoprotein and known biomarker of lung fibrosis, and NTN4 encoding Netrin-4 whose known roles include angiogenesis. The third signal may map to SLC6A6, a taurine and beta-alanine transporter gene, previously implicated in retinal, cardiac and kidney dysfunction. Conclusion:Our study has identified new associations not previously identified by previous large biobank-based studies thereby highlighting the value of utilising clinically-curated IPF case-control studies, and new genotype imputation. We present new evidence for disease-driving roles of MUC1 and of endothelial cell and vascular changes in IPF.

Progressive interstitial lung disease (ILD) leads to declining lung function and death. New therapies to treat ILD are urgently needed. Here we performed a secondary analysis of proteomic data from ten ILD cohorts across the United States, Canada, and United Kingdom. Causal mediation analysis was used to estimate the effect of plasma proteins previously linked to organ fibrosis in mechanistic studies (exposure) on survival (outcome) through lung function decline (mediator). Of 102 proteins tested in a discovery cohort (n = 1963), 47 were mediated by declining lung function. Of these 47 proteins, 7 showed sustained mediation in an independent validation cohort (n = 1172). Proteins with the strongest mediated effect were amphiregulin and integrin beta six. Sensitivity analysis showed that results were robust to unmeasured confounding. Here we provide epidemiological evidence implicating seven proteins as potentially causal of progressive ILD. These findings build upon mechanistic studies showing a causal link between these proteins and organ fibrosis, supporting their prioritization for therapeutic consideration.

Background Interstitial lung disease (ILD) represents a group of complex parenchymal conditions characterised by varying clinical trajectories. The It’s Not JUST Idiopathic Pulmonary Fibrosis Study seeks to identify genetic, proteomic and clinical biomarkers that distinguish rapidly progressive fibrotic phenotypes from stable phenotypes irrespective of aetiology. This manuscript presents baseline insights from the recruited cohort.Methods In this prospective, longitudinal study, participants with fibrotic ILDs, including idiopathic pulmonary fibrosis (IPF), fibrotic hypersensitivity pneumonitis, rheumatoid arthritis-associated ILD, asbestosis and unclassifiable ILD, were enrolled from 24 UK sites. Participants underwent comprehensive baseline evaluation including demographics, exposure history, lung function testing, 6-min walk tests, blood sampling and standardised questionnaires to assess symptoms and quality of life.Results A total of 272 participants were recruited, predominantly older white males with a smoking history. Baseline lung function showed comparable forced vital capacity (mean 89.0% predicted), diffusion of carbon monoxide (mean 57.9% predicted) and 6-min walk distance (mean 302 m) across ILD subtypes. Hypertension was the most prevalent comorbidity, affecting 40.8% of participants, with no significant differences across subtypes. Anxiety and depression were notably lower in IPF than non-IPF (4.5%; 21.0%). Previous occupational exposure was reported in 68.8% of participants, with asbestos exposure the most prevalent (36%). Bird exposure was reported by 40.4% of participants, with no significant differences across subtypes. No significant differences in health-related quality of life scores were observed across subtypes.Conclusions Despite varied aetiologies, fibrotic ILDs exhibit demographic and functional similarities, including lung function and health-related quality of life suggesting commonalities in disease mechanisms.Trial registration number NCT03670576.

Rationale: Most outcome studies in idiopathic pulmonary fibrosis (IPF) using computer-based CT analysis have focused on either phenotype or measures of disease extent in isolation. We evaluated the combination of baseline SOFIA, a UIP classifier and deep learning-based segmentation algorithms, Lung8, Air8, Vascul8, and Fibr8, for outcome prediction in a prospective IPF cohort. Methods: Participants from PROFILE with CT scans suitable for analysis were included. SOFIA provided guideline-based radiologic diagnoses and a probability score for usual interstitial pneumonia (UIP). Lung volume, airway volume, vascular volume and fibrosis volume were quantified using Lung8, Air8, Vascul8, and Fibr8, respectively. Survival analysis was performed using Cox regression. Optimal thresholds were derived for predictive metrics using ROC curve analysis. Two independent sites contributed to PROFILE; Site A was used as a derivation cohort, and Site B as a replication cohort. Results: 417 patients in PROFILE (Site A=247, Site B=170) had baseline CT scans suitable for analysis. Air8, Vascul8, and Fibr8 outputs were normalised using Lung8 (total lung volume). For Site A, SOFIA %UIP probability and normalised-Fibr8 extent (%fibrosis extent) were independently associated with increased risk of mortality (HR 1.01, p<0.0001, HR 1.04 p<0.0001, respectively, C-index=0.70). SOFIA UIP probability and normalised-Fibr8 extent had low correlation (r2=0.28 p<0.0001). A 3-stage risk score was created using thresholds for SOFIA UIP probability (22.2%, AUC 0.71) and normalised-Fibr8 extent (15.3%, AUC 0.74), optimised against disease progression stratifying patients into low (below threshold in both categories), moderate (above the threshold in one category), and high-risk (above threshold in both categories) groups. This risk score was associated with increased mortality at Site A (HR 2.21, p<0.0001, C-index 0.67) and replicated in Site B (HR 2.00, p<0.0001, C-index 0.64) and was the only metric associated with increased mortality on bivariable analysis with each threshold in both cohorts. In the whole cohort, increasing risk score was associated with increased risk of mortality controlling for %predicted FVC (HR 1.84, p<0.0001, C-index 0.69), %predicted DLco (HR 1.38, p=0.001, C-index 0.73) or the composite physiologic index (HR 1.37, p=0.002, C-index 0.72). Conclusions: CT phenotype and fibrosis quantification are independently associated with mortality in IPF and can be combined in a 3-stage risk score for predicting outcomes. Figure 1 KM Survival curve depicting survival distinctions based on the 3-stage risk score in Site A and Site B (n=417)

BACKGROUND: Leigh syndrome is a rare, progressive neurometabolic disorder caused by pathogenic variants in over 110 mitochondrial or nuclear genes. Its clinical and genetic heterogeneity creates challenges for diagnosis, care, and research. Cure Mito Foundation, a parent-led nonprofit established in 2018 to develop a gene therapy for SURF1-related Leigh syndrome, has since evolved into a global organization supporting individuals and families worldwide affected by all forms of Leigh syndrome. METHODS: This article describes the multifaceted efforts of Cure Mito Foundation to accelerate research and support for Leigh syndrome through family-led engagement and collaborative scientific partnerships. Strategies include funding the development of diverse disease models, gene therapies, drug repurposing pipelines, and a global patient registry. Emphasis is placed on co-production with affected families, sharing of biospecimens and data, and alignment with regulatory and research standards. RESULTS: The Leigh Syndrome Global Patient Registry comprises over 400 participants from 48 countries, with data made available to qualified researchers, and results shared regularly with the patient community to promote transparency and trust. Notable research accomplishments of Cure Mito include facilitating the development of multiple gene therapy candidates, patient-derived organoids and animal models, and repurposed drugs now entering early-phase trials. Cure Mito also played a key role in the launch of the Mitochondrial and Inherited Metabolic Disease Taskforce, led by the Critical Path Institute (C-Path), to integrate registry and clinical data into the Rare Disease Cures Accelerator platform. Additional efforts include community-developed educational tools, international awareness campaigns, and support programs tailored to the unique needs of Leigh syndrome families. CONCLUSIONS: Through relentless effort and dedication, the Cure Mito Foundation has shown that a small group of determined individuals can drive extraordinary change. By building a global patient registry, advancing data sharing and research, developing patient-centric education and support, and facilitating collaboration among scientists, clinicians, and families, the Foundation has created momentum toward effective treatments. With support from the Chan Zuckerberg Initiative’s Rare As One grant, Cure Mito is poised to expand its impact even further.

Lymphangioleiomyomatosis (LAM) is a rare disease of women in which TSC2 deficient 'LAM cells' with dysregulated mTOR signalling and recruited fibroblasts form nodules causing lung cysts and respiratory failure. We examine if mTOR dysregulation can induce senescence and impair the response to lung injury in LAM. The senescence markers p21, p16 and the SenMayo gene set are increased in LAM lungs and colocalise with alveolar type 2 cells. LAM models induce mTOR dependent senescence in alveolar type 2 cell organoids in vitro and in vivo. IL-6 produced by LAM cells, induces p16 and p21 in alveolar type 2 cells, inhibits epithelial wound resolution and is related to lung function in LAM patients. Rapamycin and the IL-6 receptor antagonist Tocilizumab reduce alveolar type 2 cell organoid p21 accumulation and Tocilizumab enhances epithelial wound repair. Targeting IL-6 signalling in parallel with mTOR inhibition, may reduce lung damage in LAM.

RATIONALE: There is an urgent need for reliable biomarkers to predict progression in Idiopathic Pulmonary Fibrosis (IPF). While quantitative CT metrics, automated CT phenotypes, and proteomic biomarkers have been reported, their combined prognostic utility remains unassessed. This study evaluates CT metrics and a proteomic biomarker panel in a prospective IPF cohort (PROFILE). METHODS: Participants with analysable CT scans were included. Fibrosis volume (Fibr8) as a percentage of total lung volume was quantified using a validated deep learning model. %UIP probability was evaluated using the deep learning classifier, SOFIA. Proteomic markers, with missing values <15%, were included: SPD, MMP7, PRO-C3, PRO-C4, PRO-C6, CYFRA211, and CEA. Univariate ROC-AUC curves were used to determine predictors of 12-month progression (defined as FVC decline ≥10% or death). Survival analysis was performed using Cox proportional hazards and Kaplan-Meier estimator. RESULTS: In PROFILE (n=145), on multivariable analysis, higher baseline levels of each biomarker were associated with increased mortality, adjusting for baseline ppFVC: CYFRA21-1 (HR 1.02; p<0.005), SPD (HR 1.01; p<0.005), ProC6 (HR 1.05; p=0.01), Fibr8 (HR 1.03, p=0.02), and SOFIA UIP probability (HR 2.58, P<0.005). Proteomics provided significant improvement in explaining variation of survival over imaging alone (likelihood ratio test p<0.005). Fibr8 and CYFRA21-1 had the strongest associations with 12-month progression among imaging and proteomic biomarkers. A composite risk score (CRS) was developed by fitting Fibr8 and CYFRA21-1 against 12-month progression on logistic regression: CRS=(Fibr8[asterisk]0.044) + (CYFRA21-1[asterisk]0.341). The CRS was more strongly associated with mortality and provided an improved fit over each variable in isolation when controlling for disease severity using ppFVC: CRS (HR 1.65; p<0.005; AIC 937.26), CYFRA21-1 (HR 1.21; p<0.005; AIC 947.04), Fibr8 (HR 1.04; p<0.005; AIC 942.03), ppFVC alone (HR 0.98; p<0.005; AIC 956.32). Thresholds for each biomarker, optimised against 12-month progression separated the cohort into 2 prognostically distinct groups, controlling for ppFVC: Fibr8 (n=383, 15.3%, AUC 0.69, HR 2.21), CYFRA21-1 (n=145, 2.95 ng/ml AUC 0.68, HR 1.56). The CRS improved patients’ prognostic separation over Fibr8 or CYFRA21-1 thresholds in isolation CRS (n=145, 2.09 score, AUC 0.71, HR 2.86) (Figure 1). Patients separated by CRS threshold had different ppFVC decline (p=0.01, 6.79 vs 15.69 pp/yr below/above threshold). CONCLUSION: Automated quantification of CT phenotype, fibrosis extent and protein biomarkers of epithelial injury and collagen turnover (CYFRA21-1, SPD, and ProC6) predict mortality in IPF, independently of imaging biomarkers and ppFVC. A composite risk score, combining radiological and proteomic biomarkers can be generated to provide improved prognostication in IPF.

Rationale:Higher peripheral blood monocyte count has been associated with disease progression and mortality in patients with fibrotic interstitial lung disease (fILD), but with uncertainty regarding the strength of this association and the potential impact of confounding. This study aimed to characterise the associations of clinically ascertained peripheral blood monocyte count with survival and lung function decline in patients with fILD. Methods:Patients with fILD enrolled in the prospective Canadian Registry for Pulmonary Fibrosis (CARE-PF) with baseline complete blood count were included. Monocyte counts were analysed continuously and dichotomised ≥0.6 versus <0.6×109 cells·L-1 and ≥0.95 versus <0.95×109 cells·L-1. Cox proportional hazards models, unadjusted and adjusted for age, sex, lung function, smoking and treatment, evaluated associations of monocytes with transplant-free survival. Survival analysis was repeated using the prospective PROFILE cohort. Unadjusted and adjusted linear mixed models evaluated association of monocyte count with annual decline in forced vital capacity (FVC) % predicted. Results:In 1489 patients with fILD, higher monocyte count was associated with reduced transplant-free survival in unadjusted models, but not after adjustment for relevant confounders (continuous model, HR 0.79, 95% CI 0.54-1.17; p=0.24; dichotomised at 0.6 cells·L-1, HR 0.89, 95% CI 0.72-1.10; p=0.29; and dichotomised at 0.95 cells·L-1, HR 0.93, 95% CI 0.68-1.26; p=0.62). Findings were consistent in the PROFILE external replication cohort. Monocyte count was not associated with FVC % decline in the full cohort or within fILD subtypes. Conclusions:Peripheral blood monocyte count was not associated with transplant-free survival or lung function decline in this multicentre cohort study, indicating that it is not a reliable biomarker in fILD.

Background: Lymphangioleiomyomatosis (LAM) is a rare, female-predominant, low-grade neoplasm that can lead to progressive diffuse cystic lung disease. Pregnancy has been associated with accelerated disease progression and increased risk of spontaneous pneumothoraces in women with LAM. Sirolimus is the current first line treatment for LAM and has demonstrated benefit in stabilizing lung function decline and reducing the risk of spontaneous pneumothoraces; however, there is unclear evidence regarding the risk benefit ratio of the use of sirolimus during pregnancy. Methods: We queried physicians across the LAM Clinic Network and obtained information regarding demographics, baseline characteristics, LAM related complications, lung function, sirolimus dosing, and pregnancy outcomes in women with LAM who took sirolimus during pregnancy. Results: We identified 9 women with LAM who had used sirolimus during pregnancy for a total of 12 pregnancies. All women had sporadic LAM with moderate lung function impairment prior to pregnancy: median FEV1 56% predicted, median DLCO 43% predicted, and 2 patients on supplemental oxygen. Sirolimus was used throughout pregnancy in most instances (10/12 (83.3%)). Sirolimus was held prior to pregnancy in 2 patients; both these patients experienced a spontaneous pneumothorax in the first trimester and resumed sirolimus in the second trimester with no further pneumothoraces during the rest of the pregnancy. The median sirolimus dose during pregnancy was 1 (range 1-2) mg daily with a median trough level of 2.95 (range: 1 – 6.7) ng/ml. Ten pregnancies (83.3%) resulted in live births, 1 pregnancy was terminated due to molar pregnancy, and 1 pregnancy resulted in miscarriage. Spirometry and DLCO were available in 8 pregnancies and tended to be stable (Figure 1). None of the 10 pregnancies that resulted in live births reported any fetal health issues. Seven out of these 10 children (70%) were breastfed with continued sirolimus use. At the time of submission of this report, the median age of these children is 3.5 years (range: 1 – 6.5 years) with no reported adverse health outcomes from sirolimus exposure during pregnancy. Conclusions: The use of sirolimus during pregnancy in women with LAM appears to be safe for both mother and fetus and might help reduce the pregnancy associated risks of accelerated lung function decline and spontaneous pneumothoraces.

Background Idiopathic pulmonary fibrosis (IPF) is characterised by damage to the epithelial layer, closely associated with the alveolar basement membrane (BM). We aimed to investigate how type IV collagen (COL4) in the BM changes with the progression of IPF. Methods COL4 synthesis (PRO-C4) was detected in blood by the nordicPRO-C4 biomarker in patients with IPF from the two prospective, multicentre, observational, longitudinal cohorts, pulmonary fibrosis biomarker (PFBIO) and prospective observation of fibrosis in the lung clinical endpoints (PROFILE). PRO-C4 trajectories over 12 months were compared between progressors and non-progressors by linear mixed effects regression models. Rate of change in PRO-C4 and lung function were compared by Bayesian bivariate longitudinal models. Cox proportional hazards models analysed baseline PRO-C4 and 3 years mortality. COL4 staining in IPF and non-IPF lungs was evaluated by immunohistochemistry. Results In PFBIO and PROFILE, 51/220 (23.2%) and 221/459 (48.1%) patients, respectively, had progressive disease at 12 months. Longitudinal PRO-C4 levels were higher in progressors versus non-progressors (average differences: PFBIO 21.5% (95% CI 3.4% to 42.9%, p=0.0184); PROFILE 10.9% (95% CI 0.8% to 22.1%; p=0.0340). Monthly rate of change in PRO-C4 was steeper in non-survivors versus survivors (mean difference up to 3.12% (95% CI 0.35% to 5.91%)) and was inversely correlated with the change in lung function. High baseline PRO-C4 was associated with increased mortality risk in PFBIO (HR 2.55 (95% CI 1.27 to 5.12), p=0.0083). COL4 staining was higher in IPF versus non-IPF lung but was less obvious in end-stage tissue. Conclusions High and increasing serological PRO-C4 levels were prognostic for progression in two independent IPF cohorts. This study suggests that COL4 synthesis assessed by PRO-C4 is a pathologically relevant biomarker of alveolar BM repair in IPF.

Abstract Background Genetic mitochondrial diseases are a major challenge in modern medicine. These impact ~ 1:4,000 individuals and there are currently no effective therapies. Leigh syndrome is the most common pediatric presentation of mitochondrial disease. In humans, patients are often treated with antioxidants, vitamins, and strategies targeting energetics. The vitamin-E related compound vatiquinone (EPI-743, α-tocotrienol quinone) has been the subject of at least 19 clinical trials in the US since 2012, but the effects of vatiquinone on an animal model of mitochondrial disease have not yet been reported. Here, assessed the impact of vatiquinone in cellular assays and animal models of mitochondrial disease. Methods The efficacy of vatiquinone in vitro was assessed using human fibroblasts and HEK293 cells treated with the ferroptosis inducers RSL3 and BSO + Fe(III)Citrate, the mitochondrial oxidative stress inducer paraquat, and the electron transport chain complex I inhibitor rotenone. The therapeutic potential of vatiquinone in vivo was assessed using the tamoxifen-induced mouse model for GPX4 deficiency and the Ndufs4 knockout mouse model of Leigh syndrome. Results Vatiquinone robustly prevented death in cultured cells induced by RSL3 or BSO/iron, but had no effect on paraquat induced cell death. Vatiquinone had no impact on disease onset, progression, or survival in either the tamoxifen-inducible GPX4 deficient model or the Ndufs4(-/-) mouse model, though the drug may have reduced seizure risk. Conclusions Vatiquinone prevents ferroptosis, but fails to attenuate cell death induced by paraquat or rotenone and provided no significant benefit to survival in two mouse models of disease. Vatiquinone may prevent seizures in the Ndufs4(-/-) model. Our findings are consistent with recent press statements regarding clinical trial results and have implications for drug trial design and reporting in patients with rare diseases.

Rationale Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease driven by nodules containing TSC2-/- ‘LAM cells’ and recruited LAM associated fibroblasts (LAFs). Although rapamycin reduces lung function loss, some patients continue to decline meaning additional therapies are needed.Objectives To investigate how the LAM nodule environment affects LAM cell proliferation and the response to rapamycin.Methods Changes in advanced LAM were identified using shotgun proteomics and immunohistochemistry in tissue from carefully phenotyped patients. Genes potentially associated with rapamycin insensitivity of cells grown on LAF-derived extracellular matrix were identified by RNA sequencing and validated using repurposed pharmacologic inhibitors.Main Results More advanced disease was associated with increasing nodules adjacent to lung cysts and greater decline in forced expiratory volume in 1 sec (FEV1) when treated with rapamycin (p=0.005). In late-stage LAM, proteomics identified upregulation of pathways associated with accumulation of activated fibroblasts, including extracellular matrix deposition, glucose metabolism and the actin cytoskeleton. Picrosirius red staining and immunohistochemistry confirmed deposition of extracellular matrix within LAM nodules. The growth of TSC2-/- model LAM cells was increased on LAF-derived extracellular matrix (LAF ECM), and incompletely supressed by rapamycin (p<0.0001). RNA sequencing of cells grown on LAF ECM identified upregulation of pathways driving cell cycle control, transcription and metabolism in cells. Tractable, pro-proliferative, rapamycin insensitive genes included CDK7 , GAS6 and PLAU. Repurposed inhibitors of these pathways inhibited LAM cell proliferation and enhanced the anti-proliferative effect of rapamycin.Conclusions Extracellular matrix deposited by LAM associated fibroblasts upregulates expression of genes which potentially blunt the response to rapamycin, but offer additional therapeutic opportunities for patients with established LAM.### Competing Interest StatementThe authors have declared no competing interest.

"Lymphangioleiomyomatosis: No Longer Ultra-Rare." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp.

Background Pulmonary fibrosis results from alveolar injury, leading to extracellular matrix remodelling and impaired lung function. This study aimed to classify patients with pulmonary fibrosis according to blood biomarkers to differentiate distinct disease patterns, known as endotypes. Methods In this cluster analysis, we first classified patients from the PROFILE study, a multicentre, prospective, observational cohort of individuals with incident idiopathic pulmonary fibrosis or non-specific interstitial pneumonia in the UK (Nottingham University Hospitals, Nottingham; and Royal Brompton Hospital, London). 13 blood biomarkers representing extracellular matrix remodelling, epithelial stress, and thrombosis were measured by ELISA in the PROFILE study. We classified patients by unsupervised consensus clustering. To evaluate generalisability, a machine learning classifier trained on biomarker signatures derived from consensus clustering was applied to a replication dataset from the Australian Idiopathic Pulmonary Fibrosis Registry (AIPFR). Biomarker associations with mortality and change in percentage of predicted forced vital capacity (FVC%) were assessed, adjusting for age, gender, baseline FVC%, and antifibrotic treatment and steroid treatment before and after baseline. Mortality risk associated with the clusters in the PROFILE cohort was evaluated with Cox proportional hazards models, and mixed-effects models were used to analyse how clustering was associated with longitudinal FVC% in the PROFILE and AIPFR cohorts. Findings 455 of 580 participants from the PROFILE study (348 [76%] men and 107 [24%] women; mean age 72·4 years [SD 8·3]) were included in the analysis. Within this group, three clusters were identified based on blood biomarkers. A basement membrane collagen (BM) cluster (n=248 [55%]) showed high concentrations of PRO-C4, PRO-C28, C3M, and C6M, whereas an epithelial injury (EI) cluster (n=109 [24%]) showed high concentrations of MMP-7, SP-D, CYFRA211, CA19-9, and CA-125. The third cluster (crosslinked fibrin [XF] cluster; n=98 [22%]) had high concentrations of X-FIB. In the replication dataset (117 of 833 patients from AIPFR; 87 [74%] men and 30 [26%] women; mean age 72·9 years [SD 7·9]), we identified the same three clusters (BM cluster, n=93 [79%]; EI cluster, n=8 [7%]; XF cluster, n=16 [14%]). These clusters showed similarities with clusters in the PROFILE dataset regarding blood biomarkers and phenotypic signatures. In the PROFILE dataset, the EI and XF clusters were associated with increased mortality risk compared with the BM cluster (EI vs BM: adjusted hazard ratio [HR] 1·88 [95% CI 1·42–2·49], p<0·0001; XF vs BM: adjusted HR 1·53 [1·13–2·06], p=0·0058). The EI cluster showed the greatest annual FVC% decline, followed by the BM and XF clusters. A similar FVC% decline pattern was observed in these clusters in the AIPFR replication dataset. Interpretation Blood biomarker clustering in pulmonary fibrosis identified three distinct blood biomarker signatures associated with lung function and prognosis, suggesting unique pulmonary fibrosis biomarker patterns. These findings support the presence of pulmonary fibrosis endotypes with the potential to guide targeted therapy development. Funding None.

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 FEV 1 and DL CO at first assessment were reduced to 77 and 63% predicted respectively and were similar to patients with sporadic LAM. During follow-up, FEV 1 fell by a mean of 81 ml/year and DL CO 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 FEV 1 fell by 3 ml/year and DL CO 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.