Spontaneous pneumothoraces during pregnancy in patients with lymphangioleiomyomatosis.

Patients with lymphangioleiomyomatosis (LAM) are prone to developing spontaneous pneumothoraces (SPs). We aimed to characterize the burden of SPs during pregnancy in LAM, using a web-based survey. Among the 50 respondents, 12 (24%) had never been pregnant and 38 (76%) were pregnant at least once, resulting in a total of 80 pregnancies. Respiratory symptoms during pregnancy led to the diagnosis of LAM in 34% of (13/38) patients, with SPs being the presenting manifestation in most of these patients (10/13, 77%). Eleven of the 38 pregnant patients (29%) experienced at least one SP during pregnancy. The majority of the SPs (∼60%) occurred during the second trimester. Our study provides valuable insights regarding the risk, burden, and timing of pregnancy-related SPs in patients with LAM that would be useful for the LAM community.

Single-cell multiomic analysis identifies a HOX-PBX gene network regulating the survival of lymphangioleiomyomatosis cells.

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease that predominantly affects women. LAM cells carry TSC1/TSC2 mutations, causing mTORC1 hyperactivation and uncontrolled cell growth. mTORC1 inhibitors stabilize lung function; however, sustained efficacy requires long-term administration, and some patients fail to tolerate or respond to therapy. Although the genetic basis of LAM is known, mechanisms underlying LAM pathogenesis remain elusive. We integrated single-cell RNA sequencing and single-nuclei ATAC-seq of LAM lungs to construct a gene regulatory network controlling the transcriptional program of LAM cells. We identified activation of uterine-specific HOX-PBX transcriptional programs in pulmonary LAMCORE cells as regulators of cell survival depending upon HOXD11-PBX1 dimerization. Accordingly, blockage of HOXD11-PBX1 dimerization by HXR9 suppressed LAM cell survival in vitro and in vivo. PBX1 regulated STAT1/3, increased the expression of antiapoptotic genes, and promoted LAM cell survival in vitro. The HOX-PBX gene network provides promising targets for treatment of LAM/TSC mTORC1-hyperactive cancers.

Upregulation of acid ceramidase contributes to tumor progression in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is characterized by multisystem, low-grade neoplasia involving the lung, kidneys, brain, and heart. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease affecting almost exclusively women. TSC and LAM are both caused by mutations in TSC1 and TSC2 that result in mTORC1 hyperactivation. Here, we report that single-cell RNA sequencing of LAM lungs identified activation of genes in the sphingolipid biosynthesis pathway. Accordingly, the expression of acid ceramidase (ASAH1) and dihydroceramide desaturase (DEGS1), key enzymes controlling sphingolipid and ceramide metabolism, was significantly increased in TSC2-null cells. TSC2 negatively regulated the biosynthesis of tumorigenic sphingolipids, and suppression of ASAH1 by shRNA or the inhibitor ARN14976 (17a) resulted in markedly decreased TSC2-null cell viability. In vivo, 17a significantly decreased the growth of TSC2-null cell-derived mouse xenografts and short-term lung colonization by TSC2-null cells. Combined rapamycin and 17a treatment synergistically inhibited renal cystadenoma growth in Tsc2+/- mice, consistent with increased ASAH1 expression and activity being rapamycin insensitive. Collectively, the present study identifies rapamycin-insensitive ASAH1 upregulation in TSC2-null cells and tumors and provides evidence that targeting aberrant sphingolipid biosynthesis pathways has potential therapeutic value in mechanistic target of rapamycin complex 1-hyperactive neoplasms, including TSC and LAM.

Safety and Efficacy of Combined Resveratrol and Sirolimus in Lymphangioleiomyomatosis.

BACKGROUND: A critical need exists to develop remission-inducing therapies for lymphangioleiomyomatosis. RESEARCH QUESTION: Is the addition of resveratrol safe and more efficacious than sirolimus alone in patients with lymphangioleiomyomatosis? STUDY DESIGN AND METHODS: We conducted a phase 2, dose-escalating, open-label trial of resveratrol in patients with lymphangioleiomyomatosis receiving a stable regimen of sirolimus. Resveratrol was started at 250 mg/d and escalated every 8 weeks to maximum dose of 1,000 mg/d over 24 weeks. The primary outcome was ≥ 42% decline in serum vascular endothelial growth factor D (VEGF-D) levels on combined therapy compared with baseline VEGF-D levels on sirolimus. Secondary objectives included an assessment of the safety profile and the effect on lung function and health-related quality of life (HRQOL). Longitudinal change in outcome measures was assessed using linear mixed models. Adverse effects were tabulated using the National Cancer Institute's Common Terminology Criteria for Adverse Events version 4. RESULTS: Twenty-five patients with lymphangioleiomyomatosis with a median age of 51 years were enrolled. Pulmonary function parameters at study inclusion were: FEV1: median absolute, 1.72 L; 64% predicted; FVC: median absolute, 2.99 L; 96% predicted; and diffusing capacity of the lungs for carbon monoxide: median absolute, 14.68 mL/mm Hg/min; 37% predicted. The median serum VEGF-D value at baseline was 617 pg/mL. Patients entered the study with a median sirolimus dose of 2 mg/d with median trough level of 6.3 ng/mL. Despite some GI side effects, the addition of resveratrol was well tolerated. Although the primary outcome was not met, a statistically significant reduction in serum VEGF-D levels and improvement in HRQOL during the study was found. INTERPRETATION: The addition of resveratrol was safe and well tolerated in patients with lymphangioleiomyomatosis taking sirolimus and was associated with modest improvement in HRQOL. Larger controlled trials of this combination might be warranted to assess definitively the usefulness of resveratrol as an additive therapy in lymphangioleiomyomatosis. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03253913; URL: www. CLINICALTRIALS: gov.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell.

Rationale: Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.Objectives: Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.Methods: Single-cell and single-nuclei RNA sequencing on LAM (n = 4) and control (n = 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAMCORE cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.Measurements and Main Results: A unique cell type termed LAMCORE was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAMCORE cells expressing signature genes included known LAM markers such as PMEL, FIGF, CTSK, and MLANA and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic TSC2 mutations, supporting a potential uterine origin for the LAMCORE cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.Conclusions: A unique population of LAMCORE cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.

Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia.

Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.

Estrogen activates pyruvate kinase M2 and increases the growth of TSC2-deficient cells.

Lymphangioleiomyomatosis (LAM) is a devastating lung disease caused by inactivating gene mutations in either TSC1 or TSC2 that result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). As LAM occurs predominantly in women during their reproductive age and is exacerbated by pregnancy, the female hormonal environment, and in particular estrogen, is implicated in LAM pathogenesis and progression. However, detailed underlying molecular mechanisms are not well understood. In this study, utilizing human pulmonary LAM specimens and cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we tested the hypothesis that estrogen promotes the growth of mTORC1-hyperactive cells through pyruvate kinase M2 (PKM2). Estrogen increased the phosphorylation of PKM2 at Ser37 and induced the nuclear translocation of phospho-PKM2. The estrogen receptor antagonist Faslodex reversed these effects. Restoration of TSC2 inhibited the phosphorylation of PKM2 in an mTORC1 inhibitor-insensitive manner. Finally, accumulation of phosphorylated PKM2 was evident in pulmonary nodule from LAM patients. Together, our data suggest that female predominance of LAM might be at least in part attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. Targeting metabolic regulators of PKM2 might have therapeutic benefits for women with LAM and other female-specific neoplasms.

Eight novel variants in the SLC34A2 gene in pulmonary alveolar microlithiasis.

BACKGROUND: Pulmonary alveolar microlithiasis (PAM) is caused by genetic variants in the SLC34A2 gene, which encodes the sodium-dependent phosphate transport protein 2B (NaPi-2b). PAM is characterised by deposition of calcium phosphate concretions (microliths) in the alveoli leading to pulmonary dysfunction. The variant spectrum of SLC34A2 has not been well investigated and it is not yet known whether a genotype-phenotype correlation exists. METHODS: We collected DNA from 14 patients with PAM and four relatives, and analysed the coding regions of SLC34A2 by direct DNA sequencing. To determine the phenotype characteristics, clinical data were collected and a severity score was created for each variant, based on type and localisation within the protein. RESULTS: We identified eight novel allelic variants of SLC34A2 in 14 patients with PAM. Four of these were nonsense variants, three were missense and one was a splice site variant. One patient was heterozygous for two different variants and all other patients were homozygous. Four patients were asymptomatic and 10 patients were symptomatic. The severity of the disease was associated with the variant severity. CONCLUSIONS: Our findings support a significant role for SLC34A2 in PAM and expand the variant spectrum of the disease. Thus, SLC34A2 variants were detected in all patients and eight novel allelic variants were discovered. An association between disease severity and the severity of the variants was found; however, this needs to be investigated in larger patient populations.

Spontaneous pneumothorax and air travel in Pulmonary Langerhans cell histiocytosis: A patient survey.

BACKGROUND: The optimal approach for management of spontaneous pneumothoraces (SPs) and the safety of air travel in patients with pulmonary Langerhans cell histiocytosis (PLCH) are not well established. METHODS: Patients with PLCH were recruited from the Rare Lung Diseases Clinic Network and the Histiocytosis Association, and surveyed about disease manifestations and safety of air travel. RESULTS: A total of 94 patients completed the survey. Median age at diagnosis of PLCH was 40 years (range: 15-67 years). Average interval between symptom onset and diagnosis was 2.9 years (range: -4 to 31 years). Twenty-two patients (23%) had at least one SP, of which 14 (64%) had at least one additional SP that showed either an ipsilateral recurrence (10 patients; 45%) or a contralateral recurrence (8 patients; 36%). Mean age at the time of first SP was 29 years. SP was the presenting manifestation that led to the diagnosis of PLCH in 19% of patients, typically after the second episode. Surgical pleurodesis reduced the recurrence rate of SP by half in comparison with conservative management (29% vs. 65%, p = 0.025). Two patients experienced an episode of SP during air travel, consistent with an air travel-related pneumothorax rate of 2.4% per patient and 0.27% per flight. CONCLUSIONS: SP is a common manifestation of PLCH, can be seen in approximately one-fourth of the patients, and has a high recurrence risk. Surgical pleurodesis leads to a substantial reduction in the SP recurrence risk. The risk of an air travel-related SP in patients with PLCH is about 2-3 per thousand flights. TRIAL REGISTRY CLINICALTRIALS.GOV: NCT03052101.

Tuberin Regulates Prostaglandin Receptor-Mediated Viability, via Rheb, in mTORC1-Hyperactive Cells.

Tuberous sclerosis complex (TSC) is a tumor-suppressor syndrome affecting multiple organs, including the brain, skin, kidneys, heart, and lungs. TSC is associated with mutations in TSC1 or TSC2, resulting in hyperactivation of mTOR complex 1 (mTORC1). Clinical trials demonstrate that mTORC1 inhibitors decrease tumor volume and stabilize lung function in TSC patients; however, mTOR inhibitors are cytostatic not cytocidal, and long-term benefits and toxicities are uncertain. Previously, we identified rapamycin-insensitive upregulation of cyclooxygenase 2 (PTGS2/COX2) and prostaglandin E2 (PGE2) production in TSC2-deficient cells and postulated that the action of excess PGE2 and its cognate receptors (EP) contributes to cell survival. In this study, we identify upregulation of EP3 (PTGER3) expression in TSC2-deficient cells, TSC renal angiomyolipomas, lymphangioleiomyomatosis lung nodules, and epileptic brain tubers. TSC2 negatively regulated EP3 expression via Rheb in a rapamycin-insensitive manner. The EP3 antagonist, L-798106, selectively suppressed the viability of TSC2-deficient cells in vitro and decreased the lung colonization of TSC2-deficient cells. Collectively, these data reveal a novel function of TSC2 and Rheb in the regulation of EP3 expression and cell viability.Implications: Therapeutic targeting of an aberrant PGE2-EP3 signaling axis may have therapeutic benefit for TSC patients and for other mTOR-hyperactive neoplasms. Mol Cancer Res; 15(10); 1318-30. ©2017 AACR.

A Phase II Clinical Trial of an Aromatase Inhibitor for Postmenopausal Women with Lymphangioleiomyomatosis.

RATIONALE: Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease that predominantly affects women and can worsen with pregnancy, estrogen treatment, and the menstrual cycle, suggesting an important role for estrogen in disease pathogenesis. OBJECTIVES: To assess the efficacy and safety of the aromatase inhibitor letrozole in the treatment of LAM. METHODS: Seventeen postmenopausal women with LAM were enrolled in this phase II trial and randomized to receive letrozole 2.5 mg daily (n = 9) or placebo (n = 8) for a period of 12 months. Five patients in each group were also taking sirolimus at baseline and remained on the drug throughout the treatment period. Lung function, exercise capacity, quality of life, and serum vascular endothelial growth factor D (VEGF-D) were measured at baseline and at 3-month intervals. RESULTS: Fifteen patients completed the study. Two patients withdrew. There were no differences in adverse events in the letrozole and placebo groups. The target enrollment of 25 patients per arm was not met, so the efficacy of letrozole could not be assessed as planned. After adjusting for sirolimus use, we found that the rate of change in FEV1 for all subjects was -3 ± 3 ml/mo (P = 0.4), and for serum VEGF-D, the rate of change was -0.024 ± 0.009 pg/ml/mo (P = 0.015), showing a steeper decline in the letrozole group (-0.029 ± 0.013; P = 0.025). All patients who were taking sirolimus had a reduction in VEGF-D levels from baseline to the last visit, compared with only half of the patients who were not taking sirolimus. In a post hoc analysis, eight matched letrozole-treated-placebo-treated pairs were constructed, six of which demonstrated better FEV1 improvement for the letrozole-treated patients. CONCLUSIONS: Letrozole treatment appears to be safe and well tolerated in postmenopausal patients with LAM, including those taking sirolimus. Enrollment in this trial was compromised by the publication of an effective treatment (sirolimus) in the same month as the study opened, resulting in limited power to detect treatment effects. Post hoc matched pairs exploration studies provide tentative support for additional studies of letrozole in LAM. Considering the reduced rate of lung function decline in postmenopausal patients, future studies will likely require enhanced study designs, such as selective enrollment of those with prognostic biomarkers predictive of decline. Clinical trial registered with www.clinicaltrials.gov (NCT01353209).

Spontaneous Pneumothoraces in Patients with Birt-Hogg-Dubé Syndrome.

RATIONALE: Spontaneous pneumothorax is a common complication of Birt-Hogg-Dubé syndrome (BHD). OBJECTIVES: The optimal approach to treatment and prevention of BHD-associated spontaneous pneumothorax, and to advising patients with BHD regarding risk of pneumothorax associated with air travel, is not well established. METHODS: Patients with BHD were recruited from the Rare Lung Diseases Clinic Network and the BHD Foundation and surveyed about disease manifestations and air travel experiences. RESULTS: A total of 104 patients completed the survey. The average age at diagnosis was 47 years, with an average delay from first symptoms of 13 years. Pulmonary cysts were the most frequent phenotypic manifestation of BHD, present in 85% of patients. Spontaneous pneumothorax was the presenting manifestation that led to the diagnosis of BHD in 65% of patients, typically after the second episode (mean, 2.4 episodes). Seventy-nine (76%) of 104 patients had at least one spontaneous pneumothorax during their lifetime, and 82% had multiple pneumothoraces. Among patients with multiple pneumothoraces, 73% had an ipsilateral recurrence, and 48% had a subsequent contralateral spontaneous pneumothorax following a sentinel event. The mean ages at first and second pneumothoraces were 36.5 years (range, 14-63 yr) and 37 years (range, 20-55 yr), respectively. The average number of spontaneous pneumothoraces experienced by patients with a sentinel pneumothorax was 3.6. Pleurodesis was generally performed after the second (mean, 2.4) ipsilateral pneumothorax and reduced the ipsilateral recurrence rate by half. A total of 11 episodes of spontaneous pneumothorax occurred among eight patients either during or within the 24-hour period following air travel, consistent with an air travel-related pneumothorax rate of 8% per patient and 0.12% per flight. Prior pleurodesis reduced the occurrence of a subsequent flight-related pneumothorax. CONCLUSIONS: Spontaneous pneumothorax is an important, recurrent manifestation of pulmonary involvement in patients with BHD, and pleurodesis should be considered following the initial pneumothorax to reduce the risk of recurrent episodes. In general, in patients with BHD, pneumothorax occurs in about 1-2 per 1,000 flights, and the risk is lower among patients with a history of prior pleurodesis.

Shared Decision-Making Tool for Self-Management of Home Therapies for Patients With Cystic Fibrosis.

Objective: Patients with cystic fibrosis (CF) undertake time-consuming programs of home therapies. Our objective was to develop a tool to help CF patients prioritize personal goals for some of these treatments. We describe the development and results of initial evaluation of this shared decision-making tool. Methods: Multicriteria decision-making method to develop a shared decision-making tool that integrates patient's values and perceptions of treatment impact on functionality/sense of well-being. Treatment efficacy data obtained through comprehensive review of English language literature and Cochrane reviews. Field study of 21 patients was performed to assess acceptability of the approach, understandability of the tool, and to determine whether there was sufficient patient-to-patient variability in treatment goals and patient preferences to make use of a personalized tool worthwhile. Results: Patients found the tool easy to understand and felt engaged as active participants in their care. The tool was responsive to variations in patient preferences. Priority scores were calculated (0-1.0 ± SD). Patients' most important treatment goals for improving lung health included improving breathing function (0.27 ± 0.11), improving functionality/sense of well-being (0.24 ± 0.13), preventing lung infection (0.21 ± 0.08), minimizing time to complete treatments (0.16 ± 0.12), and minimizing cost (0.11 ± 0.09). Conclusions: A shared decision-making tool that integrates patients' values and best evidence is feasible and could result in improved patient engagement in their own care.