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

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

Long-term efficacy and safety of sirolimus therapy in patients with lymphangioleiomyomatosis.

BACKGROUND: Sirolimus has been confirmed to be effective for lymphangioleiomyomatosis (LAM), a rare multisystem neoplastic disease in women. The long-term effects of sirolimus treatment for LAM, however, are largely unknown. We aimed to analyze the long-term efficacy and safety of sirolimus therapy for LAM with 4-year follow-up. METHODS: In total, 142 sporadic LAM patients who took sirolimus for 1-4 years were retrospectively enrolled for this analysis. The variables used for analysis included pulmonary function tests, arterial blood gas analysis, 6-min walking distance (6MWD), St. George's Respiratory Questionnaires (SGRQ) and serum vascular endothelial growth factor-D (VEGF-D) levels before and after the initiation of sirolimus therapy. The rates of change (slope) in those variables were calculated, and adverse events were also analyzed. RESULTS: In total, 122, 83, 60 and 32 patients out of 142 were followed for 1, 2, 3 and 4 years respectively. Sirolimus treatment improved the change rate in forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) compared with the data before treatment (FEV1, - 10 ± 15 vs. - 178 ± 36 ml/y, P <  0.001 and FVC, 54 ± 22 vs.-72 ± 68 ml/y, P < 0.05). In comparison to the baseline measurements, significant improvements were observed in FEV1 at the first year; FVC at 1-2 years; arterial oxygen levels, 6MWD, and SGRQ at 1-3 years; and VEGF-D at 1-4 years. Overall, all variables stabilized or improved during the 4 years of observation. Adverse events related to sirolimus were mild. CONCLUSION: Sirolimus therapy is effective at improving or stabilizing pulmonary function, oxygen levels, exercise capacity, and quality of life in patients with LAM for up to 4 years. VEGF-D is maintained at a lower level for 4 years after treatment. Adverse events related to sirolimus were mild.

Alterations in Polyamine Metabolism in Patients With Lymphangioleiomyomatosis and Tuberous Sclerosis Complex 2-Deficient Cells.

BACKGROUND: Lymphangioleiomyomatosis (LAM), a destructive lung disease that affects primarily women, is caused by loss-of-function mutations in TSC1 or TSC2, leading to hyperactivation of mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Rapamycin (sirolimus) treatment suppresses mTORC1 but also induces autophagy, which promotes the survival of TSC2-deficient cells. Based on the hypothesis that simultaneous inhibition of mTORC1 and autophagy would limit the availability of critical nutrients and inhibit LAM cells, we conducted a phase 1 clinical trial of sirolimus and hydroxychloroquine for LAM. Here, we report the analyses of plasma metabolomic profiles from the clinical trial. METHODS: We analyzed the plasma metabolome in samples obtained before, during, and after 6 months of treatment with sirolimus and hydroxychloroquine, using univariate statistical models and machine learning approaches. Metabolites and metabolic pathways were validated in TSC2-deficient cells derived from patients with LAM. Single-cell RNA-Seq was employed to assess metabolic enzymes in an early-passage culture from an LAM lung. RESULTS: Metabolomic profiling revealed changes in polyamine metabolism during treatment, with 5'-methylthioadenosine and arginine among the most highly upregulated metabolites. Similar findings were observed in TSC2-deficient cells derived from patients with LAM. Single-cell transcriptomic profiling of primary LAM cultured cells revealed that mTORC1 inhibition upregulated key enzymes in the polyamine metabolism pathway, including adenosylmethionine decarboxylase 1. CONCLUSIONS: Our data demonstrate that polyamine metabolic pathways are targeted by the combination of rapamycin and hydroxychloroquine, leading to upregulation of 5'-methylthioadenosine and arginine in the plasma of patients with LAM and in TSC2-deficient cells derived from a patient with LAM upon treatment with this drug combination. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01687179; URL: www.clinicaltrials.gov. Partners Human Research Committee, protocol No. 2012P000669.

Potential biomarkers of kaposiform lymphangiomatosis.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) has recently been distinguished as a novel subtype of generalized lymphatic anomaly (GLA), and is characterized by foci of spindle endothelial cells amid a background of malformed lymphatic channels. The etiology of these diseases remains unknown and diagnosis is confounded by their similar clinical findings. This study aimed to clarify differences in the clinical findings and plasma cytokine profiles of GLA and KLA patients. PROCEDURE: Clinical features data of GLA and KLA patients were obtained from a national survey. Differences in clinical findings, plasma levels of cytokines, and survival were analyzed. Plasma was obtained from healthy controls and GLA and KLA patients. Thirty-six angiogenic and lymphangiogenic factors were evaluated for cytokine concentration. RESULTS: Twenty-one patients with GLA and 11 with KLA were recruited. Mediastinal masses, hemorrhagic pericardial and pleural effusion, coagulation disorders, and thrombocytopenia were more frequent in KLA than in GLA. KLA had a significantly poorer outcome than GLA (P = 0.044). Soluble VEGFR3, angiopoietin 2, HGF, soluble HER2, tenascin C, and soluble HGFR levels were higher in KLA. Notably, soluble VEGFR3 and angiopoietin 2 levels were approximately 10-fold higher than those of other molecules measured. However, soluble VEGFR1 and soluble TIE2 were lower in KLA than in GLA and the controls. CONCLUSIONS: Patients with KLA have an unfavorable prognosis and serious symptoms (hemorrhagic pleural effusion and coagulation disorders). Our data indicate that eight angiogenic cytokines might be potential biomarkers of KLA.

Serum endostatin levels are associated with diffusion capacity and with tuberous sclerosis- associated lymphangioleiomyomatosis.

Endostatin is a naturally occurring collagen fragment with anti-angiogenic properties. We investigated the association between serum endostatin levels and DLCO in a cohort of patients with lymphangioleiomyomatosis (LAM). Associations of endostatin levels to clinical features of LAM were explored using logistic regression models. Endostatin levels were associated with DLCO and were higher in subjects with TSC-associated LAM compared to sporadic LAM. These data suggest that endostatin could be a predictive biomarker of decline in DLCO and that germline mutational inactivation of the TSC1 or TSC2 gene is associated with higher endostatin levels. These findings could offer novel insights into the pathogenesis of LAM.

Serum vascular endothelial growth factor-D as a diagnostic and therapeutic biomarker for lymphangioleiomyomatosis.

BACKGROUND: In lymphangioleiomyomatosis (LAM), tuberous sclerosis gene mutations activate the mechanistic target of the rapamycin pathway, resulting in vascular endothelial growth factor-D (VEGF-D) overproduction. While the utility of serum VEGF-D testing for the diagnosis of LAM is outlined in ATS/JRS LAM Guidelines, the assay has not been fully validated for Asian populations. Our aims were to validate serum VEGF-D testing in Japan, by directly comparing measurements in Japan and the U.S., determining the diagnostic cut-off for serum VEGF-D levels among the Japanese women with typical thin walled cystic change on CT, and determining the performance of VEGF-D as a prognostic biomarker. SUBJECTS AND METHODS: We determined serum VEGF-D levels from 108 LAM patients, 14 disease controls, and 51 healthy volunteers from the Japanese population. Measurements of 61 LAM patients were compared to those from the principal VEGF-D laboratory in the U.S at Cincinnati Children's Hospital Medical Center. We correlated baseline serum VEGF-D levels with baseline and longitudinal clinical data to determine how pregnancy, sirolimus or gonadotrophin-releasing hormone (GnRH) agonists influence serum VEGF-D levels. RESULTS: Serum VEGF-D measurements in Japan and the U.S. were very similar. Baseline serum VEGF-D levels effectively distinguished LAM from other diseases and healthy volunteers at a cut-off level of 645 pg/ml and were diagnostically specific at 800 pg/ml, consistent with the recommendations of the ATS/JRS LAM Guidelines. Baseline serum VEGF-D correlated negatively with the DLco baseline % predicted and with the annual decrease in DLco % predicted. There was no significant association between baseline serum VEGF-D level and the outcomes of death or transplant. Serum VEGF-D levels markedly decreased during treatment with sirolimus, but not with GnRH analogues. Serum VEGF-D levels of most LAM patients did not increase over time, and neither pregnancy nor menopause significantly modulated serum VEGF-D levels. CONCLUSIONS: Serum VEGF-D is a useful diagnostic and therapeutic biomarker for LAM. Satisfactory precision and international inter-laboratory agreement of the clinical assay support VEGF-D recommendations in the ATS/JRS LAM Guidelines for the Japanese population.

Concentration of Serum Vascular Endothelial Growth Factor (VEGF-D) and Its Correlation with Functional and Clinical Parameters in Patients with Lymphangioleiomyomatosis from a Brazilian Reference Center.

INTRODUCTION: Serum vascular endothelial growth factor-D (VEGF-D) is a lymphangiogenic growth factor that is considered a valuable tool in the diagnosis of lymphangioleiomyomatosis (LAM). Previous studies have reported a wide variability in VEGF-D serum levels in LAM patients and it seems to be associated with pulmonary impairment and lymphatic involvement. METHODS: We conducted a cross-sectional study from 2009 to 2017 that evaluated VEGF-D serum levels in a cohort of LAM patients who were never treated with mTOR inhibitors and compared them to healthy age-matched volunteers. Clinical and functional parameters were assessed and correlated with their respective serum VEGF-D levels. RESULTS: One hundred and four patients were included in the analysis. Serum VEGF-D levels were higher in LAM patients compared to healthy controls: 796 (404-1588) versus 162 (117-232) pg/mL, respectively (p < 0.001). Patients with tuberous sclerosis complex-LAM, TSC-LAM (20%), had higher levels of VEGF-D when compared to patients with sporadic LAM (80%) [1005 (641-2732) vs. 772 (370-1383), p = 0.05]. Serum VEGF-D levels were weakly correlated with DLCO (r = - 0.26, p = 0.001) and lymphatic involvement was more frequent in those with serum VEGF-D levels equal or above 800 pg/mL (35% vs. 13%, p = 0.02). CONCLUSIONS: In LAM, serum VEGF-D is weakly associated with lung function impairment and strongly associated with lymphatic involvement. VEGF-D is validated for use in Brazilian patients with LAM whose characteristics must be accounted for when evaluating their serum VEGF-D levels.

Dysregulation of the endothelin pathway in lymphangioleiomyomatosis with no direct effect on cell proliferation and migration.

LAM is a rare low-grade metastasizing lung neoplasm. Inhibitors of mTOR improve clinical outcome of LAM patients by preventing loss of lung function. Nevertheless, other cell targets may be of interest for drug development. Therefore, we explored the potential role of EDN1 (endothelin) in LAM. We report an increased endothelin blood level in LAM patients as well as EDN1 overexpression and EDN1 receptor downregulation in LAM-derived primary cells and in TSC2NEG cells mutated in TSC2. We evidenced EDN pathway dysregulation based on EDN1, EDNRA, EDNRB and ARRB1 mRNA expression in LAM-derived primary cells. We showed overexpression of EDN1 and ARRB1 mRNAs in TSC2NEG cells; these cells lost their ability to respond to stimulation by endothelin. We analyzed the effects of endothelin receptor antagonists alone or in combination with rapamycin, an mTOR inhibitor, on proliferation and migration of LAM cells. Rapamycin treatment of TSC2NEG cells significantly reduced cell proliferation or migration, while none of the tested inhibitors of EDN receptors impaired these functions. We showed that TSC2NEG cells have acquired a transformed phenotype as showed by their ability to grow as spheroids in semi-solid medium and that unlike endothelin receptors antagonists, rapamycin reduced anchorage-independent cell growth and prevented expansion of TSC2NEG spheroids.

Efficacy of sirolimus for the prevention of recurrent pneumothorax in patients with lymphangioleiomyomatosis: a case series.

Pneumothorax is one of the most common symptoms in patients with lymphangioleiomyomatosis (LAM). However, current management strategies for patients with LAM who present with recurrent pneumothorax remain inadequate. Here, we describe the successful prevention of recurrent pneumothorax by sirolimus treatment in five women with LAM. Before sirolimus treatment, all patients had received supplemental oxygen support, repeated chest tube drainage, or surgeries for management of the recurrent pneumothorax. Sirolimus treatment was initiated when the pneumothorax was completely resolved, and no patient developed pneumothorax during treatment. Moreover, they exhibited a significantly improved subjective quality of life, increased exercise capacity, and mild adverse effects such as mucositis, irregular menstruation, and delayed wound healing. On discontinuation of sirolimus or in the event that the plasma sirolimus level was markedly low, pneumothorax tended to relapse. The findings from these cases provide valuable insights that will aid in the improvement of treatment strategies for patients with LAM and recurrent pneumothorax.

The vitamin D binding protein axis modifies disease severity in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare disease of women. Decline in lung function is variable, making appropriate targeting of therapy difficult. We used unbiased serum proteomics to identify markers associated with outcome in LAM.101 women with LAM and 22 healthy controls were recruited from the National Centre for LAM in the UK. 152 DNA and serum samples with linked lung function and outcome data were obtained from patients in the National Heart, Lung and Blood Institute LAM Registry in the USA. Proteomic analysis was performed on a discovery cohort of 50 LAM and 20 control serum samples using a SCIEX SWATH mass spectrometric workflow. Protein levels were quantitated by ELISA and single nucleotide polymorphisms in GC (group-specific component) encoding vitamin D binding protein (VTDB) were genotyped.Proteomic analysis showed VTDB was 2.6-fold lower in LAM than controls. Serum VTDB was lower in progressive compared with stable LAM (p=0.001) and correlated with diffusing capacity of the lung for carbon monoxide (p=0.01). Median time to death or lung transplant was reduced by 46 months in those with CC genotypes at rs4588 and 38 months in those with non-A-containing haplotypes at rs7041/4588 (p=0.014 and 0.008, respectively).The VTDB axis is associated with disease severity and outcome, and GC genotype could help predict transplant-free survival in LAM.

Circulating Biomarkers From the Phase 1 Trial of Sirolimus and Autophagy Inhibition for Patients With Lymphangioleiomyomatosis.

BACKGROUND: We have previously conducted the Sirolimus and Autophagy Inhibition in LAM (SAIL) trial, a phase 1 dose-escalation study of the combination of sirolimus and hydroxychloroquine in patients with lymphangioleiomyomatosis (LAM). The goal of the present study was to analyze sera from the SAIL trial to identify novel biomarkers that could shed light into disease pathogenesis and response to therapy. METHODS: We used the DiscoveryMAP platform from Rules Based Medicine to simultaneously measure 279 analytes in sera collected at each visit from subjects enrolled in the SAIL trial. We used longitudinal regression and pathway analysis to examine analyte rate of change and corresponding effect on lung function and to identify networks and potential nodes of interest. RESULTS: A total of 222 analytes were included in the analysis. We identified 32 analytes that changed over the treatment period of the study. Pathway analysis revealed enrichment in cytokine-receptor interaction and mechanistic/mammalian target of rapamycin-related pathways, in addition to seemingly unrelated processes such as rheumatoid arthritis. Search Tool for the Retrieval of Interacting Genes/Proteins analysis identified two hubs centered around acetyl-CoA carboxylase alpha and beta and coagulation factor II. In addition, we identified vascular endothelial growth factor receptor-3 and CCL21 as molecules significantly associated with changes in FEV1 during the study period. CONCLUSIONS: We performed a large-scale analyte study in sera of women with LAM and identified potential markers that could be linked to disease pathogenesis, lung injury, and therapeutic response. These data will enable future investigation into the specific roles of these molecules in LAM. TRIAL REGISTRY: ClinicalTrials.gov; No. NCT01687179; URL: www.clinicaltrials.gov).

Clinical Course of Histologically Proven Multifocal Micronodular Pneumocyte Hyperplasia in Tuberous Sclerosis Complex: A Case Series and Comparison with Lymphangiomyomatosis.

BACKGROUND: Multifocal micronodular pneumocyte hyperplasia (MMPH) is a rare pulmonary manifestation of tuberous sclerosis complex (TSC). Because of its rarity, no previous study has described the detailed clinical course of this disease. OBJECTIVES: This study aimed to clarify the longitudinal clinical characteristics of subjects with MMPH. METHODS: Nine patients with MMPH diagnosed at Hokkaido University Hospital were retrospectively analyzed. Changes in computed tomography findings and pulmonary function were compared during the follow-up period. Serum levels of KL-6, surfactant protein (SP)-A, and SP-D were measured to clarify their potentials as blood biomarkers of the disease. Fourteen cases of lymphangiomyomatosis (LAM) were also included to compare their clinical characteristics with those of subjects with MMPH. RESULTS: Of the 9 patients, 7 were female and 2 were male. The median age at diagnosis was 43 years (range, 19-56), and all cases were diagnosed following incidental abnormal radiographic findings. During the follow-up, 1 patient died of lung cancer, but others were radiographically stable and had stable pulmonary function. Serum levels of SP-A in 5 patients (mean, 146.4 ng/mL) and SP-D in 6 patients (mean, 337.3 ng/mL) were elevated in subjects with MMPH, whereas KL-6 levels were within the reference range (mean, 230 U/mL) in all patients. Levels of SP-A and SP-D were significantly higher in subjects with MMPH than those with LAM (p < 0.05). CONCLUSIONS: Radiographic findings and pulmonary function were stable in all cases of MMPH. Serum SP-A and SP-D, but not KL-6, may be useful markers for suspicion of the presence of MMPH in patients with TSC.

Long-Term Effect of Sirolimus on Serum Vascular Endothelial Growth Factor D Levels in Patients With Lymphangioleiomyomatosis.

BACKGROUND: Sirolimus reduces serum levels of vascular endothelial growth factor D (VEGF-D); the size of chylous effusions, lymphangioleiomyomas, and angiomyolipomas; and stabilizes lung function in patients with lymphangioleiomyomatosis (LAM). METHODS: To determine whether reductions in VEGF-D levels are sustained over time, as well as parallel changes in lung function and lymphatic disease, we evaluated 25 patients with LAM and measured VEGF-D levels, lung function, and extent of lymphatic disease before and during sirolimus therapy. RESULTS: Treatment with sirolimus stabilized FEV1 and diffusion capacity for carbon monoxide (Dlco) over a period of 4.5 ± 1.6 years, caused resolution of lymphatic disease, and reduced the size of angiomyolipomas and VEGF-D levels (3,720 ± 3,020 pg/mL to 945 ± 591 pg/mL; P < .0001). Yearly changes in FEV1 % predicted and Dlco % predicted were reduced from -7.4% ± 1.4% to -0.3% ± 0.5% (P < .001) and -6.4% ± 0.9% to -0.4% ± 0.5% (P < .001), respectively. Lower VEGF-D levels correlated with sirolimus therapy (P < .001), but no significant relationship was observed between reduction in VEGF-D levels and FEV1 and Dlco during sirolimus therapy. The magnitude of VEGF-D decline was not related to the effect on lung function. Patients with lymphatic disease had higher serum VEGF-D levels, a greater reduction in VEGF-D levels, and better long-term sustained improvement in lung function during sirolimus therapy than did those without lymphatic disease. CONCLUSIONS: Sirolimus therapy stabilizes lung function over many years of therapy while producing a sustained reduction in VEGF-D levels in patients with elevated levels preceding therapy. An association was not demonstrated between the magnitude of VEGF-D decline and the beneficial effect of sirolimus on lung function. Persistent improvement in lung function was observed in patients with lymphatic disease.

CA-125 in Disease Progression and Treatment of Lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is a destructive lung disease of women caused by proliferation of neoplastic-like LAM cells, with mutations in the TSC1/2 tumor suppressor genes. Based on case reports, levels of cancer antigen 125 (CA-125), an ovarian cancer biomarker, can be elevated in patients with LAM. We hypothesized that elevated serum CA-125 levels seen in some patients with LAM were due to LAM, not other malignancies, and might respond to sirolimus treatment. METHODS: Serum CA-125 levels were measured for 241 patients at each visit. Medical records were reviewed for co-morbidities, disease progression, and response to sirolimus treatment. CA-125 expression in LAM cells was determined by using immunohistochemical analysis. RESULTS: Almost 25% of patients with LAM had at least one elevated serum CA-125 measurement. Higher serum CA-125 levels correlated with lower FEV1, premenopausal status, and pleural effusion in a multivariate model (each P < .001). Serum CA-125 levels decreased following sirolimus treatment (P = .002). CA-125 and α-smooth muscle actin were co-expressed in LAM lung nodules. CONCLUSIONS: Higher serum CA-125 levels were associated with pleural effusions and reduced pulmonary function and were decreased with sirolimus therapy. LAM cells express CA-125. Some elevated serum CA-125 levels may reflect serosal membrane involvement.

Risk factors for stomatitis in patients with lymphangioleiomyomatosis during treatment with sirolimus: A multicenter investigator-initiated prospective study.

PURPOSE: Lymphangioleiomyomatosis is a rare lung disease caused by proliferation of abnormal smooth muscle-like cells and typically occurs in premenopausal women. Sirolimus is now the first-line drug for the treatment of lymphangioleiomyomatosis. Sirolimus-induced stomatitis is the most frequent adverse event experienced during treatment. To identify risk factors, we investigated the association of stomatitis incidence with patient background data and treatment parameters, using data from the multicenter long-term sirolimus trial. METHODS: Subjects received sirolimus for 2 years at doses adjusted to maintain a trough blood level of 5 to 15 ng/mL. The incidence of stomatitis was correlated with baseline demographics, clinical characteristics, and changes in the longitudinal data. Risk factors at baseline were assessed by using univariate and multivariate analyses. RESULTS: The most frequent adverse event was stomatitis, with the cumulative rate reaching 88.9% by 9 months, higher than that reported in postrenal transplant patients. The repetition, the duration, and the severity of stomatitis events were variable among patients. We found that patients with low hemoglobin (Hb) (<14.5 g/dL) showed significantly higher incidence than those with high Hb (≥14.5 g/dL, P < .01). The cumulative rate for stomatitis incidence was significantly associated with a decrease in the mean corpuscular volume, while the Hb level was constant; thus, red blood cell count in patients increased during the study. CONCLUSIONS: Baseline Hb levels and a decrease in mean corpuscular volume during treatment were correlated with the incidence of stomatitis.

Sirolimus and Autophagy Inhibition in Lymphangioleiomyomatosis: Results of a Phase I Clinical Trial.

BACKGROUND: Animal and cellular studies support the importance of autophagy inhibition in lymphangioleiomyomatosis (LAM). In a cohort of subjects with LAM, we tested the hypothesis that treatment with sirolimus and hydroxychloroquine (an autophagy inhibitor) at two different dose levels is safe and well tolerated. Secondary end points included changes in lung function. METHODS: This 48-week, two-center phase I trial evaluated the safety of escalating oral hydroxychloroquine doses (100-200 mg) given twice a day in combination with sirolimus to eligible patients ≥ 18 years old with LAM. Subjects received combination therapy for 24 weeks followed by an observation phase without taking study drugs for an additional 24 weeks. RESULTS: Fourteen patients provided written informed consent. Thirteen were treated in cohorts of three patients each with escalating hydroxychloroquine doses (200 and 400 mg) and an extension phase at the 400-mg dose. The most common adverse events were mucositis, headache, and diarrhea. No drug-related serious adverse events were reported. Secondary end points showed improvement in lung function at 24 weeks, with a decrease in lung function at the 48-week time point. When the higher dose of hydroxychloroquine was analyzed separately, FEV1 and FVC remained stable at 48 weeks, but the 6-min walk distance showed a decrease toward baseline. CONCLUSIONS: The combination of sirolimus and hydroxychloroquine is well tolerated, with no dose-limiting adverse events observed at 200 mg twice a day. Potential effects on lung function should be explored in larger trials. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01687179; URL: www.clinicaltrials.gov.