Progression Of Lymphangioleiomyomatosis Research Articles

THU551 Glycoprotein-NMB (GPNMB) Is A Potential Lymphangioleiomyomatosis (LAM) Therapeutic Target And Biomarker

Abstract Disclosure: E. Gibbons: None. S.R. Hammes: None. Lymphangioleiomyomatosis (LAM) is a rare, progressive, destructive cystic lung disease affecting almost exclusively female-sexed individuals. Mutations in the tuberous sclerosis (TSC1 or TSC2) genes constitutively activate the mTORC1 pathway, promoting hyperproliferation of smooth muscle cells, creating small tumors throughout the lungs. These tumors, through largely unknown mechanisms, result in the formation of large cysts that replace normal alveolar space. Inhibition of mTORC1 with Sirolimus slows but does not stop LAM progression; therefore, there is a need for new LAM treatment options and biomarkers. Due to LAM’s estrogen sensitivity, female specificity, and metastatic nature, we previously proposed LAM cells originate from the uterine myometrium. We therefore designed a uterine-specific TSC2-null mouse model where all the mice generate uterine tumors characteristic of LAM and half develop lung metastases, suggesting that indeed LAM tumors may be derived from the uterus. Using bulk RNASeq analysis of uterine tissue from our mouse model, when focusing on genes regulated by estrogen and TSC2, we discovered increased expression of melanocytic markers, including Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB), a known oncogenic protein in other cancers. GPNMB was not only highly expressed in our mouse model; it was also expressed intracellularly and on the cell surface of TSC2-null cell lines and in LAM tumor cells from patient lungs and uteri. Notably, the extracellular domain of GPNMB was released from the surface of TSC2-null cell lines, suggesting that the ectodomain may also be released from TSC2-null cells in LAM patients. Here we show that, GPNMB protein and mRNA expression increased when the cells were serum starved, mimicking conditions of a tumor microenvironment. This increase was dependent on mTORC1 signaling and mRNA transcription. Knocking down GPNMB expression by siRNA had minimal effects on TSC2-null cells pro-tumorigenic functions in vitro aside from decreasing invasion. However, knocking out GPNMB in TSC2-null cells using CRISPR/Cas9 significantly decreased mouse xenograft tumor growth. Additionally, treating mice with a GPNMB antibody drug conjugate abrogated xenograft growth, highlighting GPNMB’s potential as a therapeutic target. Also, GPNMB’s ectodomain was released from the cell surface of TSC2-null cells by the proteases Adam10 and 17. Of note, the ectodomain is implicated in mediating GPNMB’s effects on tumor growth, suggesting that Adam10/17 inhibition may mitigate TSC2-null tumor growth. Importantly, the GPNMB ectodomain is a potential serum biomarker of LAM, as there was a significant increase in GPNMB ectodomain levels in patient serum as compared with healthy controls. Furthermore, serum GPNMB levels decreased in most patients after Sirolimus treatment, implicating GPNMB as a potential marker for responsiveness to treatment. Presentation: Thursday, June 15, 2023

Estradiol Augments Tumor-Induced Neutrophil Production to Promote Tumor Cell Actions in Lymphangioleiomyomatosis Models.

Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease caused by smooth muscle cell-like tumors containing tuberous sclerosis (TSC) gene mutations and found almost exclusively in females. Patient studies suggest LAM progression is estrogen dependent, an observation supported by in vivo mouse models. However, in vitro data using TSC-null cell lines demonstrate modest estradiol (E2) responses, suggesting E2 effects in vivo may involve pathways independent of direct tumor stimulation. We previously reported tumor-dependent neutrophil expansion and promotion of TSC2-null tumor growth in an E2-sensitive LAM mouse model. We therefore hypothesized that E2 stimulates tumor growth in part by promoting neutrophil production. Here we report that E2-enhanced lung colonization of TSC2-null cells is indeed dependent on neutrophils. We demonstrate that E2 induces granulopoiesis via estrogen receptor α in male and female bone marrow cultures. With our novel TSC2-null mouse myometrial cell line, we show that factors released from these cells drive E2-sensitive neutrophil production. Last, we analyzed single-cell RNA sequencing data from LAM patients and demonstrate the presence of tumor-activated neutrophils. Our data suggest a powerful positive feedback loop whereby E2 and tumor factors induce neutrophil expansion, which in turn intensifies tumor growth and production of neutrophil-stimulating factors, resulting in continued TSC2-null tumor growth.

Long-term clinical course and progression of lymphangioleiomyomatosis in a single lung transplant referral centre in Korea

We aimed to describe the clinical features of lymphangioleiomyomatosis (LAM) in Korean patients and identify factors associated with progressive disease (PD). Clinical features of 54 patients with definite or probable LAM from 2005 to 2018 were retrospectively analysed. Common features were pneumothorax (66.7%) and abdominal lymphadenopathy (50.0%). Twenty-three (42.6%) patients were initially treated with mechanistic target of rapamycin (mTOR) inhibitors. Lung transplantation (LT) was performed in 13 (24.1%) patients. Grouped based on the annual decline in forced expiratory volume in 1 s (FEV1) from baseline and LT, 36 (66.7%) patients exhibited stable disease (SD). All six deaths (11.1%) occurred in PD. Proportion of SD was higher in those treated initially with mTOR inhibitors than in those under observation (p = 0.043). Univariate analysis revealed sirolimus use, and baseline forced vital capacity, FEV1, and diffusing capacity of the lungs for carbon monoxide are associated with PD. Multivariate analysis showed that only sirolimus use (odds ratio 0.141, 95% confidence interval 0.021–0.949, p = 0.044) reduced PD. Kaplan–Meier analysis estimates overall survival of 92.0% and 74.7% at 5 and 10 years, respectively. A considerable proportion of LAM patients remain clinically stable without treatment. LT is an increasingly viable option for patients with severe lung function decline.

Soluble Immune-Related Proteins as New Candidate Serum Biomarkers for the Diagnosis and Progression of Lymphangioleiomyomatosis.

BackgroundThe goal of this study was to analyze serum from lymphangioleiomyomatosis (LAM) patients and healthy controls to identify novel biomarkers that could shed light on disease diagnosis and pathogenesis.MethodsFrom April 2017 to October 2019, qualified serum samples were obtained to explore differences in 59 immune proteins between 67 LAM patients and 49 healthy controls by the Luminex method.ResultsWe characterized 22 serum immune proteins that were differentially expressed in LAM patients compared with healthy people. Fifty-nine proteins were then classified into eight categories according to their biological function, and the results showed that LAM patients displayed significantly higher levels of growth factors (p = 0.006) and lower levels of costimulatory molecules (p = 0.008). LAG-3 was not only likely to have better predictive value than VEGF-D but also showed a significant difference between patients without elevated VEGF-D and healthy people. IL-18 was positively correlated with lung function and six-minute walk test (6MWT) distance and negatively correlated with St. George’s Respiratory Questionnaire (SGRQ) score and pulmonary artery systolic pressure (PASP), which suggested that IL-18 was related to disease severity. PD-1 was significantly different between patients with pneumothorax and/or chylothorax and those without complications.ConclusionWe performed a large-scale serum immune factor analysis of LAM. Our study provides evidence that LAG-3 may be a novel candidate serum biomarker for the diagnosis of LAM. Future independent validation in prospective studies is warranted.

Vasohibin-1 and -2 in pulmonary lymphangioleiomyomatosis (LAM) cells associated with angiogenic and prognostic factors

Lymphangioleiomyomatosis (LAM) is a rare pulmonary neoplasm, clinically associated with dyspnea and respiratory failure. Current therapeutic modalities do not necessarily reach satisfactory outcome and novel therapeutic approaches are currently warranted. Therefore, in this study, we focused on vasohibin-1 (VASH1) and -2 (VASH2); VASH1 terminated and VASH2 promoted angiogenesis. In addition, both VASH1/2 were reported to influence the progression of various human malignancies. We first performed hierarchical clustering analysis to attempt to classify 36 LAM cases into three different clusters according to immunoreactivity of VASH1/2 and other angiogenic and prognostic factors of LAM; VEGFR1/2/3, p-mTOR, p-S6, p-4EBP, ERα, PgR, MMP2, and MMP9. The cluster harboring higher angiogenic factors had higher VASH1/2 status. VASH1 was significantly positively correlated with VEGFR2, MMP9, and p-mTOR (p-value <0.05), and VASH2 with both angiogenic and prognostic factors including VEGFR1, PgR, MMP9, p-mTOR, p-S6, and p-4EBP (p-value <0.05). Subsequent PCR array of angiogenic genes demonstrated that high VASH1 mRNA was significantly positively associated with the status of SPHK1 and TYPM, lower EGF and EFNB2 (p-value <0.05), and high VASH2 mRNA negatively with MMP2 (p-value <0.05). VASH1 was considered to be up-regulated by activation of angiogenesis, whereas VASH2 could influence the angiogenesis and progression of LAM.

Differential Modulation of Matrix Metalloproteinases-2 and -7 in LAM/TSC Cells.

Matrix metalloproteinase (MMP) dysregulation is implicated in several diseases, given their involvement in extracellular matrix degradation and cell motility. In lymphangioleiomyomatosis (LAM), a pulmonary rare disease, MMP-2 and MMP-9 have been detected at high levels in serum and urine. LAM cells, characterized by a mutation in the tuberous sclerosis complex (TSC)1 or TSC2, promote cystic lung destruction. The role of MMPs in invasive and destructive LAM cell capability has not yet been fully understood. We evaluated MMP-2 and MMP-7 expression, secretion, and activity in primary LAM/TSC cells that bear a TSC2 germline mutation and an epigenetic modification and depend on epidermal growth factor (EGF) for survival. 5-azacytidine restored tuberin expression with a reduction of MMP-2 and MMP-7 levels and inhibits motility, similarly to rapamycin and anti-EGFR antibody. Both drugs reduced MMP-2 and MMP-7 secretion and activity during wound healing and decreased their expression in lung nodules of a LAM mouse model. In LAM/TSC cells, MMP-2 and MMP-7 are dependent on tuberin expression, cellular adhesion, and migration. MMPs appears sensitive to rapamycin and anti-EGFR antibody only during cellular migration. Our data indicate a complex and differential modulation of MMP-2 and MMP-7 in LAM/TSC cells, likely critical for lung parenchyma remodeling during LAM progression.

Protease activity sensors enable real-time treatment response monitoring in lymphangioleiomyomatosis.

BackgroundBiomarkers of disease progression and treatment response are urgently needed for patients with lymphangioleiomyomatosis (LAM). Activity-based nanosensors, an emerging biosensor class, detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease. Because proteases are dysregulated in LAM and may directly contribute to lung function decline, activity-based nanosensors may enable quantitative, real-time monitoring of LAM progression and treatment response. We aimed to assess the diagnostic utility of activity-based nanosensors in a pre-clinical model of pulmonary LAM.MethodsTsc2-null cells were injected intravenously into female nude mice to establish a mouse model of pulmonary LAM. A library of 14 activity-based nanosensors, designed to detect proteases across multiple catalytic classes, was administered into the lungs of LAM mice and healthy controls, urine was collected, and mass spectrometry was performed to measure nanosensor cleavage products. Mice were then treated with rapamycin and monitored with activity-based nanosensors. Machine learning was performed to distinguish diseased from healthy and treated from untreated mice.ResultsMultiple activity-based nanosensors (PP03 (cleaved by metallo, aspartic and cysteine proteases), padjusted<0.0001; PP10 (cleaved by serine, aspartic and cysteine proteases), padjusted=0.017)) were differentially cleaved in diseased and healthy lungs, enabling strong classification with a machine learning model (area under the curve (AUC) 0.95 from healthy). Within 2 days after rapamycin initiation, we observed normalisation of PP03 and PP10 cleavage, and machine learning enabled accurate classification of treatment response (AUC 0.94 from untreated).ConclusionsActivity-based nanosensors enable noninvasive, real-time monitoring of disease burden and treatment response in a pre-clinical model of LAM.

Estradiol Augments the Production and Actions of Polymorphonuclear Cells to Promote Lymphangioleiomyomatosis (LAM) Progression

Affecting almost exclusively women, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by slowly growing, estrogen-sensitive metastatic smooth muscle cell-like adenomas that result in cystic lung changes and loss of pulmonary function. LAM tumors are caused by mutations in either TSC1 or TSC2 genes that induces defective inhibition of the mTORC1 pathway, leading to increased mTORC1 activity and augmented cell proliferation. We have previously reported that estrogen ablation in our uterine-specific Tsc2 knockout mouse, which grows tumors with characteristic LAM features and lung colonization potential, effects notable regression of tumors. Thus, estrogen is required for to maintain heightened mTORC1 activity and LAM-like tumor progression. Interestingly, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol in vitro, suggesting that estradiol may act elsewhere—in mTORC1 independent manner—in vivo to promote LAM progression. Flow cytometry revealed large numbers of Ly-6Cint Ly-6Ghigh myeloid cells—polymorphonuclear cells or PMNs—in the blood and myometrial tumors of our uterine-specific Tsc2-null mice. Accordingly, we found that Tsc2-null tumors required PMNs for normal disease progression, as Gr-1 (Ly-6C/Ly-6G) depletion or inhibition of PMN recruitment reduced tumor growth. Therefore, we hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting PMN production in the bone marrow and actions in the tumor microenvironment. Using bone marrow cultures, we found that estradiol is indeed a potent inducer of PMN production. This effect occurs equally in both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ERα; knockout mice, we showed that ERα; is necessary for promoting a PMN fate for myeloid progenitors. Additionally, we have evidence implicating estrogen in the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. Overall, these data suggest that estradiol maybe facilitating crosstalk in LAM tumors, directly stimulating tumor cells while also promoting the production and actions of PMNs, which in turn promote tumor growth.

Estradiol Augments the Production and Actions of Polymorphonuclear Cells to Promote Lymphangioleiomyomatosis (LAM) Progression

Abstract Predominately affecting women of reproductive age, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by slowly growing, estrogen-sensitive metastatic smooth muscle cell-like adenomas that result in cystic lung changes and loss of pulmonary function. We have previously reported that estrogen is required to maintain LAM-like tumor progression in a uterine-specific Tsc2-knockout murine model. Interestingly, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol (E2) in vitro, suggesting that estradiol may act elsewhere to promote LAM progression. We hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting polymorphonuclear cell (PMN) production in the bone marrow and actions in the tumor microenvironment. Here, using bone marrow cultures, we demonstrate that estradiol is a potent inducer of PMN production. This effect occurs equally with both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ER_ knockout mice, we showed that ER_ is necessary for promoting a PMN fate for myeloid progenitors. While we see that the migratory and invasive capacities of TSC2-null cell lines are not augmented when stimulated with E2 directly, evidence shows estrogen promotes the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. These data provide insight into the robust effect of in vivo estrogen stimulation as estradiol may be a dual effector in LAM tumor progression and great target for anti-LAM therapeutic strategy.

The effects of total pleural covering on pneumothorax recurrence and pulmonary function in lymphangioleiomyomatosis patients without history of pleurodesis or thoracic surgeries for pneumothorax.

BackgroundTotal pleural covering (TPC) is an innovative surgical procedure in which the entire visceral pleura is wrapped with sheets of oxidized regenerated cellulose (ORC) mesh under video-assisted thoracoscopic surgery. We have previously reported that TPC could successfully prevent pneumothorax recurrence in patients with lymphangioleiomyomatosis (LAM). However, the actual efficacy and preventive effect of TPC on pneumothorax recurrence remains unclear as many LAM patients already had pleural adhesion prior to TPC that was induced by thoracic surgery and/or pleurodesis. The purpose of this study is to evaluate the effects of TPC on pneumothorax recurrence and pulmonary function in LAM patients with no history of thoracic surgeries or pleurodesis.MethodsWe retrospectively reviewed medical charts of 52 patients (60 hemithoraces) who underwent TPC at our center, from January 2003 to September 2019, as a first surgical intervention for pneumothorax.ResultsPneumothorax recurrence occurred in 12 patients [14 of 60 hemithoraces (23.3%)] during the observation period [27 months (14.7; 56.4) = median (lower; upper quartiles)]. The probability of recurrence-free hemithorax post TPC was 81.1% at 2.5 years and 64.1% at 5 years. TPC did not produce a significant decrease in either VC %predicted (pred) or FEV1/FVC. The pre- vs. post-TPC median (lower; upper quartiles) VC %pred was 85.7% (79.7; 98.0) vs. 87.2% (72.3; 95.6), P=0.535 and the FEV1/FVC was 84.6% (75.7; 89.6) vs. 83.0% (71.8; 87.0), P=0.667. Mechanistic/mammalian target of rapamycin (mTOR) inhibitors (mTORI) were subsequently initiated in 19 patients (36.5%) because of the progression of LAM. The postoperative FEV1%pred was significantly lower in patients who required mTORI than in those who did not [68.1% (57.3; 82.9) vs. 88.7% (84.6; 89.8), P=0.020]; the decline rate in FEV1%pred/year from pre to post TPC was significantly greater in LAM patients who required mTORI than in those who did not [−9.37% (−4.73; 12.9) vs. −1.94% (1.52; −4.50), P=0.029]. Postoperative complications were found in 25 of 52 hemithoraces (48.1%).ConclusionsTPC can prevent pneumothorax recurrence without causing ventilatory impairment or severe pleural symphysis in LAM patients. TPC is an effective treatment option for LAM-associated pneumothorax based on its efficacy and safety.

Mutation spectrums of TSC1 and TSC2 in Chinese women with lymphangioleiomyomatosis (LAM).

The aim of our study was to elucidate the landscapes of genetic alterations of TSC1 and TSC2 as well as other possible non-TSC1/2 in Lymphangioleiomyomatosis (LAM) patients. Sixty-one Chinese LAM patients’ clinical information was collected. Tumor biopsies and matched leukocytes from these patients were retrospectively analyzed by next generation sequencing (NGS), chromosomal microarray analysis (CMA), and multiplex ligation-dependent probe amplification (MLPA). Eighty-six TSC1/2 variants were identified in 46 of the 61 LAM patients (75.4%) in which TSC2 and TSC1 variants were 88.37% and 11.63% respectively. The 86 variants are composed of (i) 52 single nucleotide variants (SNVs) (including 30 novel variants), (ii) 23 indels (including 21deletions, and 2 insertions), (iii) a germline duplication of exon 31–42 of TSC2, (iv) a 2.68 Mb somatic duplication containing TSC2, and (v) 9 regions with copy-neutral loss of heterogeneity (CN-LOHs) present only in the LAM patients with single TSC1/2 mutations. Sixty-one non-TSC1/2 variants in 31 genes were identified in 37 LAM patients. Combined applications of different techniques are necessary to achieve maximal detection rate of TSC1/2 variants in LAM patients. Thirty novel TSC1/2 variants expands the spectrum of TSC1/2 in LAM patients. Identification of 61 non-TSC1/2 variants suggests that alternative genes might have contributed to the initiation and progression of LAM.

NO SIROLIMUS? NO PROBLEM: ALTERNATIVE MANAGEMENT OF LYMPHANGIOLEIOMYOMATOSIS DURING PREGNANCY

SESSION TITLE: Wednesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: Tuberous Sclerosis Complex (TSC) is a multisystem, autosomal-dominant condition that is associated with the development of Lymphangioleiomyomatosis (LAM). Estrogen is thought to play an important role in the pathogenesis of LAM. Increased estrogen levels during pregnancy likely contribute to the progression of LAM and the development of complications. We present a case of TSC-LAM with recurrent pneumothoraces (PTX) during pregnancy. CASE PRESENTATION: A 19-year-old gravida 1, para 0 Hispanic female with past history of TSC with known neurologic, ocular, renal, and pulmonary involvement presented with dyspnea at 24 weeks gestation to the emergency department. On presentation, patient was dyspneic but saturating well on room air, heart regular, rate and rhythm with bilateral chest expansion with decreased lung sounds on right. Chest radiograph showed right-sided PTX requiring chest tube placement. She had resolution of PTX within a few days and was discharged home. Shortly thereafter, she was readmitted with a left-sided PTX again requiring chest tube placement. Chest tube was removed after a few days with plans for pleurodesis following delivery. However, she was again readmitted now at 32nd weeks gestation with another right-sided PTX requiring chest tube placement. Due to failed clamping trials with reoccurrence of symptoms over the next two weeks, a PneumostatTM chest valve drain was placed and patient was transferred to the Obstetrics ward for continued observation. Eventually, the patient underwent a planned primary cesarean section at 38 weeks with delivery of a healthy boy. The patient did well post-operatively with plans for a future surgical pleurodesis. DISCUSSION: Pneumothoraces occur in approximately 60-70% of patients with LAM with recurrence rates over 70%. Because LAM occurs mainly in women of reproductive age, it is hypothesized that estrogen is involved in the abnormal smooth muscle proliferation. Therefore, pregnancy, a high estrogen state, is thought to increase risk of PTX in patients with LAM. While there are long term treatment options with mTOR inhibitors, such as sirolimus (pregnancy category class C), in patients with progressive lung function decline, there is a lack of literature regarding management of LAM complications during pregnancy. CONCLUSIONS: We highlight that a chest valve drain may be a viable alternative to surgical pleurodesis as a bridge to delivery in a patient concerned about surgical risk during pregnancy. This case helps solidify the need for discussion regarding the management of complications in LAM patients who become pregnant. Reference #1: Lymphangioleiomyomatosis (LAM). Breathe (Sheff). 2017;13(1):64-71. DISCLOSURES: No relevant relationships by Angela Birdwell, source=Web Response no disclosure on file for Sheila Habib; No relevant relationships by Tasnim Islam, source=Web Response Consultant relationship with Sunovion Please note: $1001 - $5000 Added 11/28/2018 by Diego Maselli Caceres, source=Web Response, value=Consulting fee Consultant relationship with Sanofi Regeneron Please note: $5001 - $20000 Added 11/28/2018 by Diego Maselli Caceres, source=Web Response, value=Consulting fee Consultant relationship with AztraZeneca Please note: $1001 - $5000 Added 03/14/2019 by Diego Maselli Caceres, source=Web Response, value=Consulting fee Consultant relationship with Novartis Please note: $1001 - $5000 Added 03/14/2019 by Diego Maselli Caceres, source=Web Response, value=Consulting fee Consultant relationship with GSK Please note: $5001 - $20000 Added 03/14/2019 by Diego Maselli Caceres, source=Web Response, value=Consulting fee No relevant relationships by Maria Velez, source=Web Response No relevant relationships by Robert Winsett, source=Web Response

Effects of sirolimus in lymphangioleiomyomatosis patients on lung cysts and pulmonary function: long-term follow-up observational study.

To determine whether sirolimus has beneficial effects on lymphangioleiomyomatosis (LAM) lung cysts in CT with long-term follow-up (FU) and to investigate whether CT is an appropriate imaging biomarker to monitor and evaluate LAM progression. In this retrospective study, 73 female patients diagnosed with definite LAM between May 2001 and June 2018 were included. Among these, 39 (53.4%) were treated with sirolimus. Quantitative and qualitative CT scoring for lung cysts (CS) were performed and compared between time points (baseline vs. FU at starting sirolimus, baseline vs. last FU, and FU at starting sirolimus vs. last FU for patients treated with sirolimus; baseline vs. last FU for patients without sirolimus). The correlation between CS at each time point and pulmonary function tests (PFTs) at each time point in the patients treated with sirolimus was also investigated. The quantitative and qualitative analyses and PFT results were compared between time points. In both quantitative and qualitative analyses, CS significantly increased from baseline to FU after starting sirolimus, and from baseline to last FU (all p < 0.05), whereas there was no significant difference between scores at the start of sirolimus vs. last in the patients treated with sirolimus. After sirolimus treatment, diffusing capacity for carbon monoxide (DLCO) was significantly increased. There were significant correlations between CS at each time point and PFT (correlation coefficient [r], - 0.383-0.935; all p < 0.001). Patients with LAM benefited from sirolimus. CT could be a useful imaging biomarker for evaluating and monitoring lung cysts in LAM. • Qualitative analysis showed a total of 15.8% to 21.1% of patients had a reduced lung cyst volume after sirolimus treatment, and in quantitative analysis, there was no significant difference in lung cyst volume between CT at the start of sirolimus therapy and the last CT. • Pulmonary function was also improved or maintained after sirolimus treatment. • Chest CT could be a useful imaging biomarker for evaluating and monitoring lung cysts in patients with lymphangioleiomyomatosis.

OR34-5 Infiltrating Neutrophils and Neutrophil Elastase (NE) Promote Tumor Growth in a Mouse Model for Lymphangioleiomyomatosis (LAM)

Lymphangioleiomyomatosis (LAM) is a rare disease occurring almost exclusively in women whereby metastatic smooth-muscle cell-like adenomas grow within the lungs, resulting in loss of pulmonary function. LAM cells contain mutations in tuberous sclerosis 1 or 2 genes (TSC1 or TSC2), which leads to heightened mammalian target of rapamycin complex1 (mTORC1) activity and cell proliferation. The LAM cell origin remains unknown; however, we reported that inactivation of the Tsc2 gene in the mouse uterus resulted in myometrial tumors with nearly all known features of LAM, including over-expression of melanocytic markers that are pathognomonic of LAM tumor cells. Approximately 50% of animals developed metastatic myometrial tumors in the lung, suggesting that LAM cells might originate from the uterine myometrium, possibly explaining its overwhelming female prevalence. Estrogen ablation by oophorectomy or aromatase inhibition resulted in nearly complete regression of Tsc2-null myometrial tumors, indicating that, even without TSC2, estradiol is required to maintain tumors. Through RNAseq analysis of genes that are both estrogen and Tsc2 mediated, we identified the serine protease Neutrophil Elastase (NE) as being highly expressed in Tsc2-null uterine tumors. NE is expressed in and secreted by granulocytic neutrophils and myeloid-derived suppressor cells (MDSCs), both of which are known to be upregulated in the blood and stroma of various cancers such as breast, lung, melanoma, and prostate. NE is thought to promote pro-tumorigenic phenotypes such as proliferation, EMT, migration, invasion, and ultimately metastasis. We hypothesize that NE from infiltrating MDSCs promotes LAM tumor progression. Using flow cytometry as well as immunofluorescence, we show increased numbers of MDSCs in the blood, uterus and lungs of Tsc2-null KO mice. Immunodepleting MDSCs results in reduced tumor growth and myometrial thickness, as does blockade of MDSC recruitment to tumors using a CXCR2 antagonist. Interestingly, treatment with the NE inhibitor sivelestat phenocopies the results from MDSC depletion, leading to reduced MDSC accumulation in the tumor site, reduced tumor growth, and less myometrial thickening. Finally, using TSC2-null cell lines, we find that NE promotes aggressive tumor cell behaviors such as migration and proliferation. Thus, our data highlights the importance of MDSCs and their product NE in LAM progression, and identifies novel molecular mediators of LAM tumor growth and invasion.

MiR‑124 targets retinoidX receptorα to reduce growth of TSC2‑deficient lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease that leads to progressive destruction of lung function. However, the mechanisms underlying the progression of LAM remain unknown. Recent studies demonstrated that miR‑124‑3p (hereinafter referred to as miR‑124) is a downregulated miRNA in tumors and it is still unclear whether miR‑124 participates in LAM. In the present study, it was revealed that miR‑124 was downregulated in LAM specimens and overexpression of miR‑124 resulted in the apoptosis of TSC2‑deficient cells via RXRα (retinoid X receptor α), while slightly influencing TSC2 wild‑type cells. Furthermore, a xenograft model demonstrated that the miR‑124/RXRα axis regulated the growth and fatty acid oxidation genes in TSC2‑null cells. Altogether, our results revealed the suppressive functions and mechanisms of miR‑124 in LAM progression, providing novel therapeutic targets for LAM treatment.

Immune Checkpoint Ligand PD-L1 Is Upregulated in Pulmonary Lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a slow-progressing metastatic disease that is driven by mutations in the tumor suppressor tuberous sclerosis complex 1/2 (TSC1/2). Rapamycin inhibits LAM cell proliferation and is the only approved treatment, but it cannot cause the regression of existing lesions and can only stabilize the disease. However, in other cancers, immunotherapies such as checkpoint blockade against PD-1 and its ligand PD-L1 have shown promise in causing tumor regression and even curing some patients. Thus, we asked whether PD-L1 has a role in LAM progression. In vitro, PD-L1 expression in murine Tsc2-null cells is unaffected by mTOR inhibition with torin but can be upregulated by IFN-γ. Using immunohistochemistry and single-cell flow cytometry, we found increased PD-L1 expression both in human lung tissue from patients with LAM and in Tsc2-null lesions in a murine model of LAM. In this model, PD-L1 is highly expressed in the lung by antigen-presenting and stromal cells, and activated T cells expressing PD-1 infiltrate the affected lung. In vivo treatment with anti-PD-1 antibody significantly prolongs mouse survival in the model of LAM. Together, these data demonstrate that PD-1/PD-L1-mediated immunosuppression may occur in LAM, and suggest new opportunities for therapeutic targeting that may provide benefits beyond those of rapamycin.

Effect of beta-agonists on LAM progression and treatment

Lymphangioleiomyomatosis (LAM), a rare disease of women, is associated with cystic lung destruction resulting from the proliferation of abnormal smooth muscle-like LAM cells with mutations in the tuberous sclerosis complex (TSC) genes TSC1 and/or TSC2 The mutant genes and encoded proteins are responsible for activation of the mechanistic target of rapamycin (mTOR), which is inhibited by sirolimus (rapamycin), a drug used to treat LAM. Patients who have LAM may also be treated with bronchodilators for asthma-like symptoms due to LAM. We observed stabilization of forced expiratory volume in 1 s over time in patients receiving sirolimus and long-acting beta-agonists with short-acting rescue inhalers compared with patients receiving only sirolimus. Because beta-agonists increase cAMP and PKA activity, we investigated effects of PKA activation on the mTOR pathway. Human skin TSC2+/- fibroblasts or LAM lung cells incubated short-term with isoproterenol (beta-agonist) showed a sirolimus-independent increase in phosphorylation of S6, a downstream effector of the mTOR pathway, and increased cell growth. Cells incubated long-term with isoproterenol, which may lead to beta-adrenergic receptor desensitization, did not show increased S6 phosphorylation. Inhibition of PKA blocked the isoproterenol effect on S6 phosphorylation. Thus, activation of PKA by beta-agonists increased phospho-S6 independent of mTOR, an effect abrogated by beta-agonist-driven receptor desensitization. In agreement, retrospective clinical data from patients with LAM suggested that a combination of bronchodilators in conjunction with sirolimus may be preferable to sirolimus alone for stabilization of pulmonary function.

Urokinase-type plasminogen activator (uPA) is critical for progression of tuberous sclerosis complex 2 (TSC2)-deficient tumors

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin-positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAM's molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1-/- and Tsc2-/- mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.

A Model of Lymphangioleiomyomatosis in a Three-Dimensional Culture System.

Lymphangioleiomyomatosis (LAM) is a tumor consisting of benign-looking neoplastic cells, but its wretched clinical outcome often resembles a malignant disease. LAM cell clusters (LCCs), unique microstructures commonly found in LAM-associated chylous effusion, are aggregates of LAM cells rimmed by lymphatic endothelium. LCCs seem to be crucial participants in the dissemination and progression of LAM. LCCs isolated from LAM-associated chylous effusion were embedded in a three-dimensional (3-D) culture gels, and then placed in a humidified CO2 incubator. During serial observations of their morphological changes, we found tube formations with lymphatic endothelial cells when LCCs settled side by side in 3-D gels. On the other hand, when LCCs were embedded separately enough to be isolated at their initial sites of settlement in the gels, each of LCCs gradually broke down, leaving a "cyst-like" hole after 7 days at the site where LCCs initially resided. Finally, we demonstrated that "cyst-like" hole formation in 3-D gels was inhibited with treatment with doxycycline or recombinant human VEGF receptor-3. We consider that our observation of this sequence in vitro illustrates how LCCs behave in vivo while enacting their important role in the progression of LAM. Our results indicate that the 3-D gel culture system for LCCs is a useful tool for exploring the effects of new therapeutic drugs under conditions when LCCs are constantly available.

Role of Hormonal Manipulations in Delaying the Progression of Lymphangioleiomyomatosis (LAM): A 16-Years Follow-Up Report after Oophorectomy

Role of Hormonal Manipulations in Delaying the Progression of Lymphangioleiomyomatosis (LAM): A 16-Years Follow-Up Report after Oophorectomy