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Review
The next breakthrough in LAM clinical trials may be their design: challenges in design and execution of future LAM clinical trials Expert Rev. Respir. Med. 9(2), 195–204 (2015)
Souheil El-Chemaly* and Elizabeth P Henske* Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, One Blackfan Circle, Karp 6th Floor, Boston, MA 02115, USA *Authors for correspondence: Tel.: +1 617 732 6869 Fax: +1 617 582 6102 [email protected] Tel.: +1 617 355 9017 Fax: +1 617 355 9016 [email protected]
The past decade has resulted in stunning progress in the pathogenesis and therapy of lymphangioleiomyomatosis (LAM), culminating in the pivotal ‘MILES’ trial, the first-ever randomized, placebo-controlled trial in LAM, demonstrating the efficacy of sirolimus in 2011. Here, we review clinical progress since 2011, focusing on new therapeutic and observational trials. These trials include the second randomized, placebo-controlled trial, a 2-year study of doxycycline effectiveness in LAM. Other clinical studies have addressed lower-dose sirolimus and treatment of pulmonary hypertension. An improved understanding of LAM pathogenesis is essential to future therapeutic breakthroughs. Critical questions that remain to be addressed include the role of estrogen and lymphangiogenesis in LAM pathogenesis and therapy, mechanisms of cystic lung destruction, the role of autophagy and pro-survival pathways in LAM cell survival. Ultimately, achieving future ‘breakthroughs’ in LAM will require continued rigorous basic and preclinical investigation, innovative clinical trial design and robust biomarkers. KEYWORDS: biomarkers . clinical trials . design . doxycycline . end points . lymphangioleiomyomatosis . mTOR inhibitors
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sirolimus . tuberous sclerosis complex
Lymphangioleiomyomatosis (LAM) is a cystic lung disease affecting primarily women, which can progress to respiratory failure and need for lung transplantation [1]. LAM can occur in a sporadic form or associated with the tuberous sclerosis complex (TSC). The progress in basic science that led to new advances in clinical LAM research has been recently reviewed by Taveira-DaSilva and Moss [2]. Our review will focus on new clinical advances in LAM since the publication of the landmark Multi-center International Lymphangioleiomyomatosis Efficacy and Safety of Sirolimus (MILES) trial in 2011, as well as the lessons learned for the design of future LAM clinical trials. New therapeutic trials & observational studies Sirolimus
It has been known for more than a decade that biallelic inactivation of the TSC genes is informahealthcare.com
10.1586/17476348.2015.1024663
associated with both the sporadic and TSCassociated forms of LAM [3,4], leading to activation of mammalian target of rapamycin complex (mTORC) 1 in LAM cells [5,6]. The MILES trial was a double-blind, placebocontrolled trial that randomly assigned women with moderately severe LAM (defined as an forced expiratory volume in 1 second (FEV1) of 50% of patients rarely requiring stopping sirolimus Changes in lung function and cyst score. Long-term safety Studies where primary end point was not change in lung function. Self prior to initiation of therapy. Low dose sirolimus: serum levels 50% of patients included hypercholesterolemia, upper respiratory tract infection, stomatitis, diarrhea, peripheral edema and acne. Sirolimus for angiomyolipomas
A Phase II trial of sirolimus for angiomyolipomas enrolled 16 patients with TSC or sporadic LAM [19]. The trial was designed to keep a steady-state level of sirolimus between informahealthcare.com
Review
3 and 6 ng/ml, which is lower than target levels in MILES [7]. Forty-one of the 48 angiomyolipomas in these patients decreased in size, most during the first year. The response rate by Response Evaluation Criteria in Solid Tumors was 50%. There was little change in pulmonary function in this singlearm study. However, for five patients who had longitudinal follow-up data prior to trial enrollment, there was a decrease in the rate of decline in lung function during the study period. Everolimus Everolimus for angiomyolipomas
A randomized, double-blind, Phase III, placebo-controlled trial of everolimus (EXIST-2) for angiomyolipomas enrolled 118 patients with at least one angiomyolipoma 3 cm or larger in longest diameter [20]. Patients received everolimus (10 mg/day) or placebo using a 2:1 assignment and were stratified by the presence of sporadic LAM. Everolimus resulted in a 50% reduction of angiomyolipoma volume in 42% of the patients, with a 50% response rate at 38 weeks of therapy. Twenty-eight (of 79) patients in the everolimus group and 18 (of 39) patients in the placebo group had a diagnosis of LAM (two sporadic LAM in the everolimus group and three sporadic LAM in the placebo group). Analysis and interpretation of pulmonary function were limited; however, the median percentage change in DLCO in the everolimus group was 3% compared to 8% in the placebo group. The median change in FEV1 was 1 and 4% in the everolimus and placebo group, respectively [20]. Adverse events were consistent with known mTOR inhibitor side effects, with stomatitis and nasopharyngitis being the most common. Fewer patients discontinued therapy in the everolimus group (4%) compared to the placebo group (10%), although the difference was not significantly different. Everolimus & lymphangiomas in LAM
In an open-label study, five women with sporadic LAM and clinically significant abdominal lymphangiomas were treated with everolimus, with serum levels titrated to 2–4 mg/l [21]. Everolimus treatment resulted in documented shrinkage of 4/5 lymphangioleiomyomas at 6 months. The fifth patient had clinical resolution of her symptoms but follow-up imaging was not performed to evaluate tumor shrinkage. In two patients with very large tumors (17.5 and 10 cm in diameter), the lesions resolved completely. No decline in lung function was observed after 6 months of therapy, and all five patients remained on therapy. In one patient, the disease recurred after treatment was temporarily discontinued. Everolimus & lung function
Since the MILES data were published, a Phase II trial of everolimus in LAM has been completed [22]. The study enrolled women with LAM with FEV1 between 50 and 80% predicted. This study was a single arm 26 weeks Phase II study of within patient dose escalation of 197
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everolimus (2.5, 5 and 10 mg daily) with the goal to study the safety and pharmacokinetics of everolimus. Secondary end points included lung function (forced vital capacity, FEV1) and measures of exercise tolerance (6 min walk test). The results of the trial are pending.
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Doxycycline & MMP blockade in LAM
The first report of possible efficacy of doxycycline in LAM was based on a single case report [23]. Two recent studies have assessed the role of doxycycline as an matrix metalloproteinase (MMP) inhibitor for the therapy of LAM [24,25]. An open-label trial by Pimenta et al. treated 41 women with LAM with 100 mg/day doxycycline for 12 months [25]. Thirty-one patients completed 12 months of treatment; these women had a mean age of 43 years, with a baseline FEV1 of 2.24 l (79% predicted) and a baseline DLCO of 17.0 ml/min/mmHg (65% predicted). Both urine and serum MMP-9 levels were markedly elevated in the LAM patients at baseline compared with a group of 10 healthy control women of similar mean age (40 years). Overall, there was a mean decrease of 70 ml in FEV1 and a mean decrease of 0.24 ml/min/mmHg in DLCO. The authors concluded that doxycycline had ‘no impact on pulmonary function decline’ based on comparison with historical rates of decline of between 75 ml and 118 ml/year [26,27], although the use of historical controls in LAM research has obvious shortcomings. An additional caveat about this doxycycline study is that 22 of the 31 patients were also receiving hormonal blockade therapy. Interesting changes in MMPs were observed in this study. The serum MMP-9 was 10-fold higher in the LAM patients (933 ng/ml in the LAM patients vs 89.60 ng/ml in the healthy controls) and the urinary MMP-9 was 50-fold higher (10,487 vs 200 pg/ml). Treatment with doxycycline did not decrease MMP-9 or VEGF-D in the serum, although MMP-9 levels in the urine declined. Thirteen patients (the ‘doxycycline responders’) showed a slight increase in FEV1 over the 12 months of therapy, from a mean of 2.31 (84% predicted) to 2.40 l (86% predicted), while the remaining 20 patients (‘doxycycline nonresponders’) had a decrease in FEV1, from a mean of 2.2 (75% predicted) to 2.0 l (70% predicted). Somewhat surprisingly, given the assumed mechanism of action of doxycycline as an MMP inhibitor, no association between MMP inhibition and the responder versus non-responder group or the rate of decline of lung function was observed. In the absence of a placebo control group, and given that the majority of the patients on this trial were receiving hormonal blockade therapy (although there was no significant difference between the responders and nonresponders in the usage of hormonal therapy), it is challenging to translate these data on doxycycline into clinical practice. The evidence of elevated serum MMP-9 levels in this study supports the prior work of several groups [28,29] that MMPs may participate in lung destruction in LAM. Correlations between MMP-9 levels and hormonal status (the majority of the patients were receiving hormonal therapy) would be of considerable interest, given that prior work [29] has 198
demonstrated that MMP expression and activity are estrogen responsive. In the second doxycycline study, Chang et al. [24] randomized 23 women with a mean FEV1 of 1.69 l (58% predicted) to doxycycline (100 mg/day for 3 months followed by 200 mg/day for 21 months) versus placebo control. The average age was 46 years; one-third were postmenopausal. A total of 17 patients completed 12 months of treatment (8 placebo and 9 doxycycline) and 15 patients completed 2 years of treatment (7 placebo and 8 doxycycline). The change in FEV1 using an intention-totreat analysis was 90.3 ml/year in the placebo group and d 123 ml/year in the doxycycline group (p = 0.35). No difference between the groups was observed at any 6-month interval (6, 12, 18 or 24 months). Twelve patients had angiomyolipomas (six placebo and six doxycycline); no change in size was observed in either group. No differences were found in dyspnea score as measured by the St. George’s respiratory questionnaire. Adverse events leading to withdrawal included four pneumothoraces (one in the doxycycline group), a fall in FEV1 >300 ml (doxycycline) and a seizure leading to discovery of a meningioma (doxycycline). Two patients in the doxycycline group had severe respiratory infections. Consistent with the study by Pimenta et al., no change in serum VEGF-D was observed. A decrease in total urinary MMP-9 was found (p = 0.03). Thus, this study failed to demonstrate the benefit of doxycycline using a randomized, placebo-controlled design. Whether a subgroup of women with mild disease would respond to doxycycline [25,30,31] is unclear. Furthermore, whether doxycycline in combination with mTOR inhibitors would have clinical benefit in LAM is unknown. Hormonal therapies in LAM
Since LAM preferentially occurs in women, and because of reports of worsening of disease during pregnancy, hormones, especially estrogen, have been suspected to play a role in disease pathogenesis [32,33]. Furthermore, in preclinical models, estrogen enhances the lung colonization of TSC2-deficient cells [34] and an estrogen inhibitor resulted in decreased LAM cell homing into the lungs [29]. Progesterone – a long accepted therapy for LAM – did not show any benefit in a retrospective analysis of women treated or not with progesterone [26]. More recently, a prospective study of gonadotropin releasing hormone at a dose of 11.25 mg intramuscularly every 3 months in premenopausal women with LAM showed no benefit from drug administration in comparison to historical controls, with continued decline in lung function [35]. The role of estrogen blockage was also evaluated in the Trial of Aromatase Inhibition in LAM trial [36]. This is a Phase II trial of an aromatase inhibitor letrozole 2.5 mg daily for 12 months compared to placebo in postmenopausal women with LAM. The aromatase enzyme is largely responsible for estrogen production in postmenopausal women by converting adrenal androgens to estrogens. The primary end point is the effect on FEV1. Results of the trial are pending. Expert Rev. Respir. Med. 9(2), (2015)
The next breakthrough in LAM clinical trials may be their design
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Abnormalities in diffusion capacity & pulmonary hypertension in LAM
Resting pulmonary hypertension occurs in 50% of patients [17]. Furthermore, at the time of initial enrollment in the national LAM registry, a decrease in diffusion capacity (which could in a subset of patients represent evidence of pulmonary hypertension) was the second most common pulmonary function abnormality after airflow obstruction [33]. The MILES trial did not show any benefit for sirolimus in halting the decline in DLCO [7], and as discussed above, long-term follow-up on sirolimus showed that gains in the stability of DLCO were lost overtime [14]. Thus, there are many unknowns regarding the therapeutic benefit of rapamycin or specific pulmonary hypertension therapies in treating pulmonary arterial hypertension in LAM. To address this knowledge gap, Cottin et al. evaluated 29 women with LAM who underwent right heart catheterization [16]. PH was defined as a mean pulmonary artery pressure >25 mmHg and pulmonary artery wedge pressure 12% and 200 ml increase in FEV1 over baseline has been associated with worse outcome in women with LAM [37]. However, the percentage of women with LAM found to have a positive response to BDs varies informahealthcare.com
Pulmonary rehabilitation in LAM
Pulmonary rehabilitation has a proven benefit in obstructive [42] and interstitial lung disease [43,44]. Similar to chronic obstructive pulmonary disease, pulmonary rehabilitation could lead to improved dyspnea, exercise tolerance and quality of life in 199
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Table 2. Current therapeutic clinical trials in LAM†. Trial name
Drug
Phase
Dose
Duration
Identifier
SAIL
Sirolimus and hydroxychloroquine
Phase I dose escalation
Sirolimus + hydroxychloroquine 200 or 400 mg daily
6 months on and 6 months off therapy
NCT01687179 [50]
SOS
Sirolimus and simvastatin
Phase I–II
Sirolimus/everolimus + simvastatin 20 mg for 2 months then 40 mg for 2 months
4 months
NCT02061397 [59]
SLAM-1
Saracatinib
Phase I dose escalation
Saracatinib 50, 125 and 175 mg
4 weeks
NCT02116712 [60]
† Source clinicaltrials.gov. LAM: Lymphangioleiomyomatosis; SAIL: Sirolimus and autophagy inhibition in LAM; SLAM-1: The tolerability of saracatinib in subjects with LAM; SOS: Safety of simvastatin in LAM and TSC.
LAM. The benefits of pulmonary rehabilitation in LAM are currently being investigated [45]. This is an observational study of a 12-week pulmonary rehabilitation program (1 h of treadmill exercise and muscle strength training). The end point is endurance time during cycle ergometry. Secondary end points are quality of life and other lung function parameters. This study aims to establish the clinical effectiveness of pulmonary rehabilitation in LAM.
Patients would receive 6 months of therapy followed by 6 months of observation off therapy to monitor if stability in lung function is maintained off study drugs. These hydroxychloroquine doses are similar to those used for the treatment of Sjogren syndrome [51] and lower than the maximum tolerated dose identified in a study of autophagy blockade in glioblastoma [52]. SRC inhibitor
Current clinical trials in LAM
Based on a series of observations in vitro and in animal models, indicating that some drugs – discussed below – offer synergistic benefits in combination with rapamycin, multiple Phase I clinical trials are evaluating the safety of different agents alone or in combination with mTOR inhibitors (TABLE 2). Simvastatin in LAM
Preclinical evidence demonstrated that simvastatin in combination with sirolimus was an effective inhibitor of lung destruction and cyst formation in an animal model of LAM [46–48]. A Phase I study designed to evaluate the safety of the combination of simvastatin and mTOR inhibition in LAM is currently underway. The study aims at evaluating the safety of the combination of simvastatin (20 mg daily) and mTOR inhibitors (either sirolimus or everolimus) for 2 months. If safety is established, simvastatin dose will be increased to 40 mg daily, as in vitro studies have shown that simvastatin had a concentration-dependent inhibitory effect on mTORC1 [46]. The doses of simvastatin used are in line with doses used for the treatment of hypercholesterolemia. Sirolimus and autophagy inhibition in LAM
Preclinical evidence showed that LAM cells are dependent on autophagy for survival [49,50]. The central hypothesis of this study is that blocking autophagy in addition to inhibition of the mTOR pathway would lead to LAM cells death. This trial evaluates the combination of sirolimus (at the same doses used in MILES) with hydroxychloroquine (200 mg once or twice daily) in a classic Phase I dose escalation study. 200
TSC2 null cells have increased Src kinase activity, which leads to increased migration and invasion of these cells in vitro and in vivo, which is significantly reduced by inhibitors of Src kinase [53,54]. Based on these findings, a Phase I dose escalation study of Src inhibition is currently underway. This is a Phase I dose escalation study designed to study the safety and tolerability of three different doses of saracatinib – a Src inhibitor – in women with LAM. A Phase I study of three different doses (50, 75 and 150 mg given orally once a day). The drug will be given daily orally for 4 weeks with an additional follow-up of 4 more weeks. The doses used in this study are comparable to doses used in oncology (175 mg) [55,56]. Preparing for the next breakthrough in LAM
The completion of two randomized, placebo-controlled therapeutic clinical trials in LAM (sirolimus and doxycycline) is an extraordinary accomplishment for the LAM patient and research community [7,24]. The clinical advances related to the proven efficacy of sirolimus in LAM contribute toward the logistical difficulties and the costs associated with clinical trials that will lead to the next breakthroughs. We believe that continued advances in the treatment of LAM are dependent on an improved understanding of disease pathogenesis. Some of the most critical basic and translational priorities include: .
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To optimize cell culture and animal models of LAM, especially ones that allow the mechanisms of estrogen’s actions and the mechanisms of lung cyst pathogenesis to be more fully understood. To identify the cell-of-origin of LAM. Expert Rev. Respir. Med. 9(2), (2015)
The next breakthrough in LAM clinical trials may be their design
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Start of therapy Prior of therapy
Observational phase
Lung function
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Non-toxic therapy
Therapy that can improve lung function Randomized controlled trials Therapy that can be stopped mTOR inhibitors Therapy for mTOR inhibitors non-responders
Time
Figure 1. Needs and potential approaches to future clinical trials in lymphangioleiomyomatosis. Future Phase II and III clinical trials could benefit from enrolling patients with known rates of decline in lung function. Observational studies where each patient is their own control could provide a foundation for future randomized controlled trials. There is an urgent need for agents: with minimal toxicity that could be taken early in the disease to stabilize lung function; that could not only stabilize but also improve lung function; that would lead to remission and therefore not require continued use; and that would benefit patients who do not respond to or cannot tolerate inhibitors of the mTOR pathways. . .
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To identify genetic, molecular and/or biochemical mechanisms that promote disease progression. To understand the role(s) of the lymphatic circulation, suspected to be the conduit for LAM cell metastasis and dissemination [57]. To understand mechanisms of VEGF-D production and function in LAM, including the possible intracellular functions of VEGF-D in lung fibroblasts [58,59]. To identify the stage at which LAM pathogenesis begins – is it during fetal or childhood development?
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From our review of published LAM clinical studies since the MILES trial, a series of observations emerge: .
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In the absence of a randomized controlled trial, knowledge of individual patient true rate of decline in lung function before initiation of therapy offers the best possible alternative to gain insight into efficacy of a drug. Studies by Ando et al. and Yao et al. [12,15] offer compelling arguments of the feasibility of such design and the important information that can be gained from fewer enrolled subjects. Acknowledging the challenge of information safety, differences in local institutional review boards and relative cost, it is critical to establish clinical coordinating centers, where information about individual patient lung function are stored and can be accessed for potential inclusion in clinical trials. Understanding LAM phenotypes is critical. In addition to each patient’s own rate of decline in lung function, knowing, whether patients with lymphatic involvement or high VEGF-D can be included in the same study as patients whose only manifestation is cystic lung disease with normal VEGF-D levels. The absence of such understanding generates
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important unanswered questions even after trials are completed. For instance, ‘Is doxycycline useful for women with LAM and mild disease?’ [25,30,31] Establishing biomarkers that are of clear clinical utility [1]. Urinary MMP-9 decreased in response to therapy with doxycycline [24,25,60]. Is it a useful marker of disease progression? This challenge highlights the importance of continued support for basic sciences in order to better understand disease pathogenesis. Understanding the sensitivity of clinical indicators, such as 6 minute walk test and CPET, as markers of disease progression, with the ultimate goal of evaluating these parameters as end points in clinical trials. Coordination and prioritization of clinical trial needs and design across the USA and worldwide. Our own view is summarized in FIGURE 1.
In closing, current therapies with mTORC1 inhibitors, such as sirolimus, appear to suppress but not eliminate LAM cells. Continuous therapy is likely required, since lung function tends to decline and angiomyolipomas re-grow when therapy is discontinued. The ultimate goal is to eliminate LAM cells before they have caused significant loss of lung function, which will require early detection and a safe, well-tolerated therapy that eradicates TSC2-deficient cells. To accomplish this and other breakthroughs will require a coordinated, multi-disciplinary and carefully prioritized matrix of basic, preclinical and clinical investigation. Expert commentary
Advances in our understanding of the pathogenesis of LAM have led to a landmark Phase III randomized controlled trial 201
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that identified sirolimus as an effective therapy for LAM. Sirolimus stabilizes lung function in women with LAM, although the benefit is not maintained when treatment is stopped. Therefore, continuous sirolimus therapy is required. Since the publication of the MILES trial, several studies have extended our understanding of the effectiveness of mTOR inhibitors and their long-term effects. In aggregate, these data confirm the efficacy of sirolimus and highlight the need for additional therapeutic options for patients who cannot tolerate long-term mTOR inhibition or are unresponsive to mTOR inhibitors. In addition, there remains an unmet need for therapies (either single agent or in combination with mTOR inhibitors) that can lead to durable ‘remission’ of LAM and therefore do not need to be used indefinitely. Several early stage clinical trials are ongoing that build upon preclinical data that support agents or combination that may lead to new therapies for LAM. A major limitation remains the challenge of conducting a clinical trial in a rare disease, especially now that effective therapy is available. Fast-tracking therapeutic development in LAM will require a coordinated effort including: the identification of subsets of patients with more rapidly progressive disease and/or disease subtypes that may respond to specific therapeutic strategies; the development of surrogate biomarkers of therapeutic efficacy; and innovative clinical trial design. Together, these will enable more efficient early-phase trials and thus streamline the prioritization of agents to be evaluated in randomized controlled trials.
LAM, including therapies that induce a true remission (and therefore continuous administration is not required). The next 4 years, from 2011 to 2015, have yielded abundant preclinical data about the fundamental mechanisms of LAM pathogenesis, and several of these preclinical studies have already been translated into ongoing Phase I clinical trials. Meanwhile, the results of two Phase II trials, focused on everolimus and aromatase inhibition, are pending. In the next 5 years, from 2015 to 2020, the highest priorities include: continued clinical and preclinical investigation to better understand disease pathogenesis, including the role of lymphangiogenesis and the reasons that a subset of women with LAM have lower VEGF-D levels; the development of biomarkers (serum biomarkers, imaging tools, genetics or clinical features) that can serve as ‘surrogates’ of therapeutic response as an adjunct to pulmonary function in early phase clinical trials; and innovative clinical trial design that will allow efficient testing for new therapeutics in LAM. These may include trials in which each patient serves as her own control (n = 1). The strengths of the LAM research community include: the motivated, informed and organized patient community; the well-established track-record of international collaboration among both preclinical and clinical LAM investigators; and a robust and rapidly growing basic and translational scientific foundation of LAM pathogenesis and therapy. Together, these critical elements will ensure continued success and progress over the next 5 years toward optimizing the therapeutic options for women with LAM.
Five-year view
Publication of the landmark MILES trial in 2011, demonstrating the efficacy of sirolimus in LAM, was a milestone in the history of LAM. It is remarkable that this trial was published just 11 years after TSC2 mutations were identified as the cause of sporadic LAM. Despite this stunning progress, there is an urgent unmet need for additional therapies for women with
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Key issues. .
Lymphangioleiomyomatosis is a cystic lung disease caused by mutations in the TSC1 or TSC2 genes, which leads to activation of the mTOR pathway.
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The MILES trial has established the effectiveness of sirolimus (an mTOR inhibitor) to halt lung function decline.
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There is a need for continuous use of sirolimus; therefore, additional therapies are needed.
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Therapies are needed for women who do not respond or cannot tolerate mTOR inhibitors.
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Therapies that can be discontinued.
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The difficulty in conducting a Phase II–III clinical trial in a rare disease with an existing therapy.
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Future drugs or combination of drugs could perhaps be first tested in patients with known rates of decline in lung function. This would allow each patient in essence to serve as their own control, reducing the numbers needed to enroll.
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Biomarkers for disease activity are needed that would allow their use as end points, therefore reducing the numbers needed to enroll.
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The next breakthrough in LAM clinical trials may be their design
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1.
El-Chemaly S, Henske EP. Towards personalised therapy for lymphangioleiomyomatosis: lessons from cancer. Eur Respir Rev 2014;23:30-5
13.
Taveira-DaSilva AM, Hathaway O, Stylianou M, Moss J. Changes in lung function and chylous effusions in patients with lymphangioleiomyomatosis treated with sirolimus. Ann Intern Med 2011;154: 797-805. W-292-793
14.
Yao J, Taveira-DaSilva AM, Jones AM, et al. Sustained effects of sirolimus on lung function and cystic lung lesions in lymphangioleiomyomatosis. Am J Respir Crit Care Med 2014;190:1273-82
15.
Yao J, Taveira-DaSilva AM, Jones AM, et al. Sustained effects of sirolimus on lung function and cystic lung lesions in lymphangioleiomyomatosis. Am J Respir Crit Care Med 2014;190(11):1273-82
16.
Cottin V, Harari S, Humbert M, et al. Pulmonary hypertension in lymphangioleiomyomatosis: characteristics in 20 patients. Eur Respir J 2012;40:630-40
17.
Taveira-DaSilva AM, Hathaway OM, Sachdev V, et al. Pulmonary artery pressure in lymphangioleiomyomatosis: an echocardiographic study. Chest 2007;132:1573-8
18.
McCormack FX, Inoue Y, Moss J, et al. Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N Engl J Med 2011;364:1595-606 Seyama K, Kumasaka T, Souma S, et al. Vascular endothelial growth factor-D is increased in serum of patients with lymphangioleiomyomatosis. Lymphat Res Biol 2006;4:143-52
2.
Taveira-DaSilva AM, Moss J. Optimizing treatments for lymphangioleiomyomatosis. Expert Rev Respir Med 2012;6:267-76
3.
Carsillo T, Astrinidis A, Henske EP. Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc Natl Acad Sci USA 2000;97:6085-90
4.
5.
6.
7.
8.
9.
10.
11.
12.
Yu J, Astrinidis A, Henske EP. Chromosome 16 loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis. Am J Respir Crit Care Med 2001;164:1537-40 Henske EP, McCormack FX. Lymphangioleiomyomatosis - a wolf in sheep’s clothing. J Clin Invest 2012;122: 3807-16 Goncharova EA, Goncharov DA, Eszterhas A, et al. Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). J Biol Chem 2002;277:30958-67
24.
Chang WY, Cane JL, Kumaran M, et al. A 2-year randomised placebo-controlled trial of doxycycline for lymphangioleiomyomatosis. Eur Respir J 2014;43:1114-23
25.
Pimenta SP, Baldi BG, Kairalla RA, Carvalho CR. Doxycycline use in patients with lymphangioleiomyomatosis: biomarkers and pulmonary function response. J Bras Pneumol 2013;39:5-15
26.
Taveira-DaSilva AM, Stylianou MP, Hedin CJ, et al. Decline in lung function in patients with lymphangioleiomyomatosis treated with or without progesterone. Chest 2004;126:1867-74
27.
Johnson SR, Whale CI, Hubbard RB, et al. Survival and disease progression in UK patients with lymphangioleiomyomatosis. Thorax 2004;59:800-3
28.
Glassberg MK, Elliot SJ, Fritz J, et al. Activation of the estrogen receptor contributes to the progression of pulmonary lymphangioleiomyomatosis via matrix metalloproteinase-induced cell invasiveness. J Clin Endocrinol Metab 2008;93:1625-33
Zafar MA, McCormack FX, Rahman S, et al. Pulmonary vascular shunts in exercise-intolerant patients with lymphangioleiomyomatosis. Am J Respir Crit Care Med 2013;188:1167-70
29.
Li C, Zhou X, Sun Y, et al. Faslodex inhibits estradiol-induced extracellular matrix dynamics and lung metastasis in a model of lymphangioleiomyomatosis. Am J Respir Cell Mol Biol 2013;49:135-42
19.
Davies DM, de Vries PJ, Johnson SR, et al. Sirolimus therapy for angiomyolipoma in tuberous sclerosis and sporadic lymphangioleiomyomatosis: a phase 2 trial. Clin Cancer Res 2011;17:4071-81
30.
Baldi BG, Ribeiro Carvalho CR. Doxycycline in lymphangioleiomyomatosis: not all questions are answered. Eur Respir J 2014;43:1536-7
31.
20.
Bissler JJ, Kingswood JC, Radzikowska E, et al. Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, double-blind, placebo-controlled trial. Lancet 2013;381:817-24
Johnson SR, Chang WY, Tattersfield AE, et al. Doxycycline in lymphangioleiomyomatosis: not all questions are answered. Eur Respir J 2014;43:1538
32.
Brunelli A, Catalini G, Fianchini A. Pregnancy exacerbating unsuspected mediastinal lymphangioleiomyomatosis and chylothorax. Int J Gynaecol Obstet 1996;52:289-90
33.
Ryu JH, Moss J, Beck GJ, et al. The NHLBI lymphangioleiomyomatosis registry: characteristics of 230 patients at enrollment. Am J Respir Crit Care Med 2006;173: 105-11
34.
Yu JJ, Robb VA, Morrison TA, et al. Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells. Proc Natl Acad Sci USA 2009;106:2635-40
35.
Harari S, Cassandro R, Chiodini I, et al. Effect of a gonadotrophin-releasing hormone analogue on lung function in
Young LR, Vandyke R, Gulleman PM, et al. Serum vascular endothelial growth factor-D prospectively distinguishes lymphangioleiomyomatosis from other diseases. Chest 2010;138:674-81 Young LR, Lee HS, Inoue Y, et al. Serum VEGF-D concentration as a biomarker of lymphangioleiomyomatosis severity and treatment response: a prospective analysis of the Multicenter International Lymphangioleiomyomatosis Efficacy of Sirolimus (MILES) trial. Lancet Respir Med 2013;1:445-52 El-Chemaly S, Goldberg HJ, Glanville AR. Should mammalian target of rapamycin inhibitors be stopped in women with lymphangioleiomyomatosis awaiting lung transplantation? Expert Rev Respir Med 2014;8:657-60 Ando K, Kurihara M, Kataoka H, et al. Efficacy and safety of low-dose sirolimus for
informahealthcare.com
monitoring for MMPs. N Engl J Med 2006;354:2621-2
treatment of lymphangioleiomyomatosis. Respir Investig 2013;51:175-83
References
Review
21.
22.
23.
Mohammadieh AM, Bowler SD, Silverstone EJ, et al. Everolimus treatment of abdominal lymphangioleiomyoma in five women with sporadic lymphangioleiomyomatosis. Med J Aust 2013;199:121-3 A study to determine the effectiveness of escalating doses of RAD001 (Everolimus) in patients with lymphangioleiomyomatosis. Available from: https://clinicaltrials.gov/ct2/ show/NCT01059318 Moses MA, Harper J, Folkman J. Doxycycline treatment for lymphangioleiomyomatosis with urinary
203
Review
El-Chemaly & Henske
Expert Review of Respiratory Medicine Downloaded from informahealthcare.com by Nanyang Technological University on 04/25/15 For personal use only.
lymphangioleiomyomatosis. Chest 2008;133:448-54 36.
Trial of aromatase inhibition in lymphangioleiomyomatosis (TRAIL). Available from: https://clinicaltrials.gov/ct2/ show/NCT01353209
37.
Taveira-DaSilva AM, Steagall WK, Rabel A, et al. Reversible airflow obstruction in lymphangioleiomyomatosis. Chest 2009;136:1596-603
38.
39.
40.
Nebulized or inhaled albuterol for lymphangioleiomyomatosis. Available from: https://clinicaltrials.gov/ct2/show/ NCT01799538 Baldi BG, Albuquerque AL, Pimenta SP, et al. Exercise performance and dynamic hyperinflation in lymphangioleiomyomatosis. Am J Respir Crit Care Med 2012;186:341-8 Baldi BG, de Albuquerque AL, Pimenta SP, et al. A pilot study assessing the effect of bronchodilator on dynamic hyperinflation in LAM. Respir Med 2013;107:1773-80
42.
Puhan MA, Lareau SC. Evidence-based outcomes from pulmonary rehabilitation in the chronic obstructive pulmonary disease patient. Clin Chest Med 2014;35:295-301
44.
45.
46.
47.
Yen KT, Putzke JD, Staats BA, Burger CD. The prevalence of acute response to bronchodilator in pulmonary lymphangioleiomyomatosis. Respirology 2005;10:643-8
41.
43.
https://clinicaltrials.gov/ct2/show/ NCT02009241
Dowman L, Hill CJ, Holland AE. Pulmonary rehabilitation for interstitial lung disease. Cochrane Database Syst Rev 2014;10:CD006322 Rochester CL, Fairburn C, Crouch RH. Pulmonary rehabilitation for respiratory disorders other than chronic obstructive pulmonary disease. Clin Chest Med 2014;35:369-89 Pulmonary Rehabilitation in Lymphangioleiomyomatosis. Available from:
204
Atochina-Vasserman EN, Goncharov DA, Volgina AV, et al. Statins in Lymphangioleiomyomatosis. Simvastatin and atorvastatin induce differential effects on tuberous sclerosis complex 2-null cell growth and signaling. Am J Respir Cell Mol Biol 2013;49:704-9 Goncharova EA, Goncharov DA, Fehrenbach M, et al. Prevention of alveolar destruction and airspace enlargement in a mouse model of pulmonary lymphangioleiomyomatosis (LAM). Sci Transl Med 2012;4:154ra134
48.
Safety of Simvastatin in LAM and TSC (SOS). Available from: https://clinicaltrials. gov/ct2/show/NCT02061397
49.
Parkhitko A, Myachina F, Morrison TA, et al. Tumorigenesis in tuberous sclerosis complex is autophagy and p62/ sequestosome 1 (SQSTM1)-dependent. Proc Natl Acad Sci USA 2011;108:12455-60
50.
Safety study of sirolimus and hydroxychloroquine in women with lymphangioleiomyomatosis (SAIL). Available from: https://clinicaltrials.gov/ct2/show/ NCT01687179
51.
Gottenberg JE, Ravaud P, Puechal X, et al. Effects of hydroxychloroquine on symptomatic improvement in primary Sjogren syndrome: the JOQUER randomized clinical trial. JAMA 2014;312: 249-58
52.
53.
Rosenfeld MR, Ye X, Supko JG, et al. A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy 2014;10:1359-68
lymphangioleiomyomatosis. Cancer Res 2014;74:1996-2005 54.
The tolerability of saracatinib in subjects with lymphangioleiomyomatosis (LAM) (SLAM-1). Available from: https:// clinicaltrials.gov/ct2/show/NCT02116712
55.
McNeish IA, Ledermann JA, Webber L, et al. A randomised, placebo-controlled trial of weekly paclitaxel and saracatinib (AZD0530) in platinum-resistant ovarian, fallopian tube or primary peritoneal cancer dagger. Ann Oncol 2014;25:1988-95
56.
Molina JR, Foster NR, Reungwetwattana T, et al. A phase II trial of the Src-kinase inhibitor saracatinib after four cycles of chemotherapy for patients with extensive stage small cell lung cancer: NCCTG trial N-0621. Lung Cancer 2014;85:245-50
57.
Seyama K, Kumasaka T, Kurihara M, et al. Lymphangioleiomyomatosis: a disease involving the lymphatic system. Lymphat Res Biol 2010;8:21-31
58.
Cui Y, Osorio JC, Risquez C, et al. Transforming growth factor-beta1 downregulates vascular endothelial growth factor-D expression in human lung fibroblasts via the Jun NH2-terminal kinase signaling pathway. Mol Med 2014;20:120-34
59.
El-Chemaly S, Pacheco-Rodriguez G, Malide D, et al. Nuclear localization of vascular endothelial growth factor-D and regulation of c-Myc-dependent transcripts in human lung fibroblasts. Am J Respir Cell Mol Biol 2014;51:34-42
60.
Pimenta SP, Baldi BG, Acencio MM, et al. Doxycycline use in patients with lymphangioleiomyomatosis: safety and efficacy in metalloproteinase blockade. J Bras Pneumol 2011;37:424-30
Tyryshkin A, Bhattacharya A, Eissa NT. SRC kinase is a novel therapeutic target in
Expert Rev. Respir. Med. 9(2), (2015)
Review
The next breakthrough in LAM clinical trials may be their design: challenges in design and execution of future LAM clinical trials Expert Rev. Respir. Med. 9(2), 195–204 (2015)
Souheil El-Chemaly* and Elizabeth P Henske* Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, One Blackfan Circle, Karp 6th Floor, Boston, MA 02115, USA *Authors for correspondence: Tel.: +1 617 732 6869 Fax: +1 617 582 6102 [email protected] Tel.: +1 617 355 9017 Fax: +1 617 355 9016 [email protected]
The past decade has resulted in stunning progress in the pathogenesis and therapy of lymphangioleiomyomatosis (LAM), culminating in the pivotal ‘MILES’ trial, the first-ever randomized, placebo-controlled trial in LAM, demonstrating the efficacy of sirolimus in 2011. Here, we review clinical progress since 2011, focusing on new therapeutic and observational trials. These trials include the second randomized, placebo-controlled trial, a 2-year study of doxycycline effectiveness in LAM. Other clinical studies have addressed lower-dose sirolimus and treatment of pulmonary hypertension. An improved understanding of LAM pathogenesis is essential to future therapeutic breakthroughs. Critical questions that remain to be addressed include the role of estrogen and lymphangiogenesis in LAM pathogenesis and therapy, mechanisms of cystic lung destruction, the role of autophagy and pro-survival pathways in LAM cell survival. Ultimately, achieving future ‘breakthroughs’ in LAM will require continued rigorous basic and preclinical investigation, innovative clinical trial design and robust biomarkers. KEYWORDS: biomarkers . clinical trials . design . doxycycline . end points . lymphangioleiomyomatosis . mTOR inhibitors
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Rheb
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sirolimus . tuberous sclerosis complex
Lymphangioleiomyomatosis (LAM) is a cystic lung disease affecting primarily women, which can progress to respiratory failure and need for lung transplantation [1]. LAM can occur in a sporadic form or associated with the tuberous sclerosis complex (TSC). The progress in basic science that led to new advances in clinical LAM research has been recently reviewed by Taveira-DaSilva and Moss [2]. Our review will focus on new clinical advances in LAM since the publication of the landmark Multi-center International Lymphangioleiomyomatosis Efficacy and Safety of Sirolimus (MILES) trial in 2011, as well as the lessons learned for the design of future LAM clinical trials. New therapeutic trials & observational studies Sirolimus
It has been known for more than a decade that biallelic inactivation of the TSC genes is informahealthcare.com
10.1586/17476348.2015.1024663
associated with both the sporadic and TSCassociated forms of LAM [3,4], leading to activation of mammalian target of rapamycin complex (mTORC) 1 in LAM cells [5,6]. The MILES trial was a double-blind, placebocontrolled trial that randomly assigned women with moderately severe LAM (defined as an forced expiratory volume in 1 second (FEV1) of 50% of patients rarely requiring stopping sirolimus Changes in lung function and cyst score. Long-term safety Studies where primary end point was not change in lung function. Self prior to initiation of therapy. Low dose sirolimus: serum levels 50% of patients included hypercholesterolemia, upper respiratory tract infection, stomatitis, diarrhea, peripheral edema and acne. Sirolimus for angiomyolipomas
A Phase II trial of sirolimus for angiomyolipomas enrolled 16 patients with TSC or sporadic LAM [19]. The trial was designed to keep a steady-state level of sirolimus between informahealthcare.com
Review
3 and 6 ng/ml, which is lower than target levels in MILES [7]. Forty-one of the 48 angiomyolipomas in these patients decreased in size, most during the first year. The response rate by Response Evaluation Criteria in Solid Tumors was 50%. There was little change in pulmonary function in this singlearm study. However, for five patients who had longitudinal follow-up data prior to trial enrollment, there was a decrease in the rate of decline in lung function during the study period. Everolimus Everolimus for angiomyolipomas
A randomized, double-blind, Phase III, placebo-controlled trial of everolimus (EXIST-2) for angiomyolipomas enrolled 118 patients with at least one angiomyolipoma 3 cm or larger in longest diameter [20]. Patients received everolimus (10 mg/day) or placebo using a 2:1 assignment and were stratified by the presence of sporadic LAM. Everolimus resulted in a 50% reduction of angiomyolipoma volume in 42% of the patients, with a 50% response rate at 38 weeks of therapy. Twenty-eight (of 79) patients in the everolimus group and 18 (of 39) patients in the placebo group had a diagnosis of LAM (two sporadic LAM in the everolimus group and three sporadic LAM in the placebo group). Analysis and interpretation of pulmonary function were limited; however, the median percentage change in DLCO in the everolimus group was 3% compared to 8% in the placebo group. The median change in FEV1 was 1 and 4% in the everolimus and placebo group, respectively [20]. Adverse events were consistent with known mTOR inhibitor side effects, with stomatitis and nasopharyngitis being the most common. Fewer patients discontinued therapy in the everolimus group (4%) compared to the placebo group (10%), although the difference was not significantly different. Everolimus & lymphangiomas in LAM
In an open-label study, five women with sporadic LAM and clinically significant abdominal lymphangiomas were treated with everolimus, with serum levels titrated to 2–4 mg/l [21]. Everolimus treatment resulted in documented shrinkage of 4/5 lymphangioleiomyomas at 6 months. The fifth patient had clinical resolution of her symptoms but follow-up imaging was not performed to evaluate tumor shrinkage. In two patients with very large tumors (17.5 and 10 cm in diameter), the lesions resolved completely. No decline in lung function was observed after 6 months of therapy, and all five patients remained on therapy. In one patient, the disease recurred after treatment was temporarily discontinued. Everolimus & lung function
Since the MILES data were published, a Phase II trial of everolimus in LAM has been completed [22]. The study enrolled women with LAM with FEV1 between 50 and 80% predicted. This study was a single arm 26 weeks Phase II study of within patient dose escalation of 197
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everolimus (2.5, 5 and 10 mg daily) with the goal to study the safety and pharmacokinetics of everolimus. Secondary end points included lung function (forced vital capacity, FEV1) and measures of exercise tolerance (6 min walk test). The results of the trial are pending.
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Doxycycline & MMP blockade in LAM
The first report of possible efficacy of doxycycline in LAM was based on a single case report [23]. Two recent studies have assessed the role of doxycycline as an matrix metalloproteinase (MMP) inhibitor for the therapy of LAM [24,25]. An open-label trial by Pimenta et al. treated 41 women with LAM with 100 mg/day doxycycline for 12 months [25]. Thirty-one patients completed 12 months of treatment; these women had a mean age of 43 years, with a baseline FEV1 of 2.24 l (79% predicted) and a baseline DLCO of 17.0 ml/min/mmHg (65% predicted). Both urine and serum MMP-9 levels were markedly elevated in the LAM patients at baseline compared with a group of 10 healthy control women of similar mean age (40 years). Overall, there was a mean decrease of 70 ml in FEV1 and a mean decrease of 0.24 ml/min/mmHg in DLCO. The authors concluded that doxycycline had ‘no impact on pulmonary function decline’ based on comparison with historical rates of decline of between 75 ml and 118 ml/year [26,27], although the use of historical controls in LAM research has obvious shortcomings. An additional caveat about this doxycycline study is that 22 of the 31 patients were also receiving hormonal blockade therapy. Interesting changes in MMPs were observed in this study. The serum MMP-9 was 10-fold higher in the LAM patients (933 ng/ml in the LAM patients vs 89.60 ng/ml in the healthy controls) and the urinary MMP-9 was 50-fold higher (10,487 vs 200 pg/ml). Treatment with doxycycline did not decrease MMP-9 or VEGF-D in the serum, although MMP-9 levels in the urine declined. Thirteen patients (the ‘doxycycline responders’) showed a slight increase in FEV1 over the 12 months of therapy, from a mean of 2.31 (84% predicted) to 2.40 l (86% predicted), while the remaining 20 patients (‘doxycycline nonresponders’) had a decrease in FEV1, from a mean of 2.2 (75% predicted) to 2.0 l (70% predicted). Somewhat surprisingly, given the assumed mechanism of action of doxycycline as an MMP inhibitor, no association between MMP inhibition and the responder versus non-responder group or the rate of decline of lung function was observed. In the absence of a placebo control group, and given that the majority of the patients on this trial were receiving hormonal blockade therapy (although there was no significant difference between the responders and nonresponders in the usage of hormonal therapy), it is challenging to translate these data on doxycycline into clinical practice. The evidence of elevated serum MMP-9 levels in this study supports the prior work of several groups [28,29] that MMPs may participate in lung destruction in LAM. Correlations between MMP-9 levels and hormonal status (the majority of the patients were receiving hormonal therapy) would be of considerable interest, given that prior work [29] has 198
demonstrated that MMP expression and activity are estrogen responsive. In the second doxycycline study, Chang et al. [24] randomized 23 women with a mean FEV1 of 1.69 l (58% predicted) to doxycycline (100 mg/day for 3 months followed by 200 mg/day for 21 months) versus placebo control. The average age was 46 years; one-third were postmenopausal. A total of 17 patients completed 12 months of treatment (8 placebo and 9 doxycycline) and 15 patients completed 2 years of treatment (7 placebo and 8 doxycycline). The change in FEV1 using an intention-totreat analysis was 90.3 ml/year in the placebo group and d 123 ml/year in the doxycycline group (p = 0.35). No difference between the groups was observed at any 6-month interval (6, 12, 18 or 24 months). Twelve patients had angiomyolipomas (six placebo and six doxycycline); no change in size was observed in either group. No differences were found in dyspnea score as measured by the St. George’s respiratory questionnaire. Adverse events leading to withdrawal included four pneumothoraces (one in the doxycycline group), a fall in FEV1 >300 ml (doxycycline) and a seizure leading to discovery of a meningioma (doxycycline). Two patients in the doxycycline group had severe respiratory infections. Consistent with the study by Pimenta et al., no change in serum VEGF-D was observed. A decrease in total urinary MMP-9 was found (p = 0.03). Thus, this study failed to demonstrate the benefit of doxycycline using a randomized, placebo-controlled design. Whether a subgroup of women with mild disease would respond to doxycycline [25,30,31] is unclear. Furthermore, whether doxycycline in combination with mTOR inhibitors would have clinical benefit in LAM is unknown. Hormonal therapies in LAM
Since LAM preferentially occurs in women, and because of reports of worsening of disease during pregnancy, hormones, especially estrogen, have been suspected to play a role in disease pathogenesis [32,33]. Furthermore, in preclinical models, estrogen enhances the lung colonization of TSC2-deficient cells [34] and an estrogen inhibitor resulted in decreased LAM cell homing into the lungs [29]. Progesterone – a long accepted therapy for LAM – did not show any benefit in a retrospective analysis of women treated or not with progesterone [26]. More recently, a prospective study of gonadotropin releasing hormone at a dose of 11.25 mg intramuscularly every 3 months in premenopausal women with LAM showed no benefit from drug administration in comparison to historical controls, with continued decline in lung function [35]. The role of estrogen blockage was also evaluated in the Trial of Aromatase Inhibition in LAM trial [36]. This is a Phase II trial of an aromatase inhibitor letrozole 2.5 mg daily for 12 months compared to placebo in postmenopausal women with LAM. The aromatase enzyme is largely responsible for estrogen production in postmenopausal women by converting adrenal androgens to estrogens. The primary end point is the effect on FEV1. Results of the trial are pending. Expert Rev. Respir. Med. 9(2), (2015)
The next breakthrough in LAM clinical trials may be their design
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Abnormalities in diffusion capacity & pulmonary hypertension in LAM
Resting pulmonary hypertension occurs in 50% of patients [17]. Furthermore, at the time of initial enrollment in the national LAM registry, a decrease in diffusion capacity (which could in a subset of patients represent evidence of pulmonary hypertension) was the second most common pulmonary function abnormality after airflow obstruction [33]. The MILES trial did not show any benefit for sirolimus in halting the decline in DLCO [7], and as discussed above, long-term follow-up on sirolimus showed that gains in the stability of DLCO were lost overtime [14]. Thus, there are many unknowns regarding the therapeutic benefit of rapamycin or specific pulmonary hypertension therapies in treating pulmonary arterial hypertension in LAM. To address this knowledge gap, Cottin et al. evaluated 29 women with LAM who underwent right heart catheterization [16]. PH was defined as a mean pulmonary artery pressure >25 mmHg and pulmonary artery wedge pressure 12% and 200 ml increase in FEV1 over baseline has been associated with worse outcome in women with LAM [37]. However, the percentage of women with LAM found to have a positive response to BDs varies informahealthcare.com
Pulmonary rehabilitation in LAM
Pulmonary rehabilitation has a proven benefit in obstructive [42] and interstitial lung disease [43,44]. Similar to chronic obstructive pulmonary disease, pulmonary rehabilitation could lead to improved dyspnea, exercise tolerance and quality of life in 199
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Table 2. Current therapeutic clinical trials in LAM†. Trial name
Drug
Phase
Dose
Duration
Identifier
SAIL
Sirolimus and hydroxychloroquine
Phase I dose escalation
Sirolimus + hydroxychloroquine 200 or 400 mg daily
6 months on and 6 months off therapy
NCT01687179 [50]
SOS
Sirolimus and simvastatin
Phase I–II
Sirolimus/everolimus + simvastatin 20 mg for 2 months then 40 mg for 2 months
4 months
NCT02061397 [59]
SLAM-1
Saracatinib
Phase I dose escalation
Saracatinib 50, 125 and 175 mg
4 weeks
NCT02116712 [60]
† Source clinicaltrials.gov. LAM: Lymphangioleiomyomatosis; SAIL: Sirolimus and autophagy inhibition in LAM; SLAM-1: The tolerability of saracatinib in subjects with LAM; SOS: Safety of simvastatin in LAM and TSC.
LAM. The benefits of pulmonary rehabilitation in LAM are currently being investigated [45]. This is an observational study of a 12-week pulmonary rehabilitation program (1 h of treadmill exercise and muscle strength training). The end point is endurance time during cycle ergometry. Secondary end points are quality of life and other lung function parameters. This study aims to establish the clinical effectiveness of pulmonary rehabilitation in LAM.
Patients would receive 6 months of therapy followed by 6 months of observation off therapy to monitor if stability in lung function is maintained off study drugs. These hydroxychloroquine doses are similar to those used for the treatment of Sjogren syndrome [51] and lower than the maximum tolerated dose identified in a study of autophagy blockade in glioblastoma [52]. SRC inhibitor
Current clinical trials in LAM
Based on a series of observations in vitro and in animal models, indicating that some drugs – discussed below – offer synergistic benefits in combination with rapamycin, multiple Phase I clinical trials are evaluating the safety of different agents alone or in combination with mTOR inhibitors (TABLE 2). Simvastatin in LAM
Preclinical evidence demonstrated that simvastatin in combination with sirolimus was an effective inhibitor of lung destruction and cyst formation in an animal model of LAM [46–48]. A Phase I study designed to evaluate the safety of the combination of simvastatin and mTOR inhibition in LAM is currently underway. The study aims at evaluating the safety of the combination of simvastatin (20 mg daily) and mTOR inhibitors (either sirolimus or everolimus) for 2 months. If safety is established, simvastatin dose will be increased to 40 mg daily, as in vitro studies have shown that simvastatin had a concentration-dependent inhibitory effect on mTORC1 [46]. The doses of simvastatin used are in line with doses used for the treatment of hypercholesterolemia. Sirolimus and autophagy inhibition in LAM
Preclinical evidence showed that LAM cells are dependent on autophagy for survival [49,50]. The central hypothesis of this study is that blocking autophagy in addition to inhibition of the mTOR pathway would lead to LAM cells death. This trial evaluates the combination of sirolimus (at the same doses used in MILES) with hydroxychloroquine (200 mg once or twice daily) in a classic Phase I dose escalation study. 200
TSC2 null cells have increased Src kinase activity, which leads to increased migration and invasion of these cells in vitro and in vivo, which is significantly reduced by inhibitors of Src kinase [53,54]. Based on these findings, a Phase I dose escalation study of Src inhibition is currently underway. This is a Phase I dose escalation study designed to study the safety and tolerability of three different doses of saracatinib – a Src inhibitor – in women with LAM. A Phase I study of three different doses (50, 75 and 150 mg given orally once a day). The drug will be given daily orally for 4 weeks with an additional follow-up of 4 more weeks. The doses used in this study are comparable to doses used in oncology (175 mg) [55,56]. Preparing for the next breakthrough in LAM
The completion of two randomized, placebo-controlled therapeutic clinical trials in LAM (sirolimus and doxycycline) is an extraordinary accomplishment for the LAM patient and research community [7,24]. The clinical advances related to the proven efficacy of sirolimus in LAM contribute toward the logistical difficulties and the costs associated with clinical trials that will lead to the next breakthroughs. We believe that continued advances in the treatment of LAM are dependent on an improved understanding of disease pathogenesis. Some of the most critical basic and translational priorities include: .
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To optimize cell culture and animal models of LAM, especially ones that allow the mechanisms of estrogen’s actions and the mechanisms of lung cyst pathogenesis to be more fully understood. To identify the cell-of-origin of LAM. Expert Rev. Respir. Med. 9(2), (2015)
The next breakthrough in LAM clinical trials may be their design
Review
Start of therapy Prior of therapy
Observational phase
Lung function
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Non-toxic therapy
Therapy that can improve lung function Randomized controlled trials Therapy that can be stopped mTOR inhibitors Therapy for mTOR inhibitors non-responders
Time
Figure 1. Needs and potential approaches to future clinical trials in lymphangioleiomyomatosis. Future Phase II and III clinical trials could benefit from enrolling patients with known rates of decline in lung function. Observational studies where each patient is their own control could provide a foundation for future randomized controlled trials. There is an urgent need for agents: with minimal toxicity that could be taken early in the disease to stabilize lung function; that could not only stabilize but also improve lung function; that would lead to remission and therefore not require continued use; and that would benefit patients who do not respond to or cannot tolerate inhibitors of the mTOR pathways. . .
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To identify genetic, molecular and/or biochemical mechanisms that promote disease progression. To understand the role(s) of the lymphatic circulation, suspected to be the conduit for LAM cell metastasis and dissemination [57]. To understand mechanisms of VEGF-D production and function in LAM, including the possible intracellular functions of VEGF-D in lung fibroblasts [58,59]. To identify the stage at which LAM pathogenesis begins – is it during fetal or childhood development?
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From our review of published LAM clinical studies since the MILES trial, a series of observations emerge: .
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In the absence of a randomized controlled trial, knowledge of individual patient true rate of decline in lung function before initiation of therapy offers the best possible alternative to gain insight into efficacy of a drug. Studies by Ando et al. and Yao et al. [12,15] offer compelling arguments of the feasibility of such design and the important information that can be gained from fewer enrolled subjects. Acknowledging the challenge of information safety, differences in local institutional review boards and relative cost, it is critical to establish clinical coordinating centers, where information about individual patient lung function are stored and can be accessed for potential inclusion in clinical trials. Understanding LAM phenotypes is critical. In addition to each patient’s own rate of decline in lung function, knowing, whether patients with lymphatic involvement or high VEGF-D can be included in the same study as patients whose only manifestation is cystic lung disease with normal VEGF-D levels. The absence of such understanding generates
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important unanswered questions even after trials are completed. For instance, ‘Is doxycycline useful for women with LAM and mild disease?’ [25,30,31] Establishing biomarkers that are of clear clinical utility [1]. Urinary MMP-9 decreased in response to therapy with doxycycline [24,25,60]. Is it a useful marker of disease progression? This challenge highlights the importance of continued support for basic sciences in order to better understand disease pathogenesis. Understanding the sensitivity of clinical indicators, such as 6 minute walk test and CPET, as markers of disease progression, with the ultimate goal of evaluating these parameters as end points in clinical trials. Coordination and prioritization of clinical trial needs and design across the USA and worldwide. Our own view is summarized in FIGURE 1.
In closing, current therapies with mTORC1 inhibitors, such as sirolimus, appear to suppress but not eliminate LAM cells. Continuous therapy is likely required, since lung function tends to decline and angiomyolipomas re-grow when therapy is discontinued. The ultimate goal is to eliminate LAM cells before they have caused significant loss of lung function, which will require early detection and a safe, well-tolerated therapy that eradicates TSC2-deficient cells. To accomplish this and other breakthroughs will require a coordinated, multi-disciplinary and carefully prioritized matrix of basic, preclinical and clinical investigation. Expert commentary
Advances in our understanding of the pathogenesis of LAM have led to a landmark Phase III randomized controlled trial 201
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that identified sirolimus as an effective therapy for LAM. Sirolimus stabilizes lung function in women with LAM, although the benefit is not maintained when treatment is stopped. Therefore, continuous sirolimus therapy is required. Since the publication of the MILES trial, several studies have extended our understanding of the effectiveness of mTOR inhibitors and their long-term effects. In aggregate, these data confirm the efficacy of sirolimus and highlight the need for additional therapeutic options for patients who cannot tolerate long-term mTOR inhibition or are unresponsive to mTOR inhibitors. In addition, there remains an unmet need for therapies (either single agent or in combination with mTOR inhibitors) that can lead to durable ‘remission’ of LAM and therefore do not need to be used indefinitely. Several early stage clinical trials are ongoing that build upon preclinical data that support agents or combination that may lead to new therapies for LAM. A major limitation remains the challenge of conducting a clinical trial in a rare disease, especially now that effective therapy is available. Fast-tracking therapeutic development in LAM will require a coordinated effort including: the identification of subsets of patients with more rapidly progressive disease and/or disease subtypes that may respond to specific therapeutic strategies; the development of surrogate biomarkers of therapeutic efficacy; and innovative clinical trial design. Together, these will enable more efficient early-phase trials and thus streamline the prioritization of agents to be evaluated in randomized controlled trials.
LAM, including therapies that induce a true remission (and therefore continuous administration is not required). The next 4 years, from 2011 to 2015, have yielded abundant preclinical data about the fundamental mechanisms of LAM pathogenesis, and several of these preclinical studies have already been translated into ongoing Phase I clinical trials. Meanwhile, the results of two Phase II trials, focused on everolimus and aromatase inhibition, are pending. In the next 5 years, from 2015 to 2020, the highest priorities include: continued clinical and preclinical investigation to better understand disease pathogenesis, including the role of lymphangiogenesis and the reasons that a subset of women with LAM have lower VEGF-D levels; the development of biomarkers (serum biomarkers, imaging tools, genetics or clinical features) that can serve as ‘surrogates’ of therapeutic response as an adjunct to pulmonary function in early phase clinical trials; and innovative clinical trial design that will allow efficient testing for new therapeutics in LAM. These may include trials in which each patient serves as her own control (n = 1). The strengths of the LAM research community include: the motivated, informed and organized patient community; the well-established track-record of international collaboration among both preclinical and clinical LAM investigators; and a robust and rapidly growing basic and translational scientific foundation of LAM pathogenesis and therapy. Together, these critical elements will ensure continued success and progress over the next 5 years toward optimizing the therapeutic options for women with LAM.
Five-year view
Publication of the landmark MILES trial in 2011, demonstrating the efficacy of sirolimus in LAM, was a milestone in the history of LAM. It is remarkable that this trial was published just 11 years after TSC2 mutations were identified as the cause of sporadic LAM. Despite this stunning progress, there is an urgent unmet need for additional therapies for women with
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Key issues. .
Lymphangioleiomyomatosis is a cystic lung disease caused by mutations in the TSC1 or TSC2 genes, which leads to activation of the mTOR pathway.
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The MILES trial has established the effectiveness of sirolimus (an mTOR inhibitor) to halt lung function decline.
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There is a need for continuous use of sirolimus; therefore, additional therapies are needed.
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Therapies are needed for women who do not respond or cannot tolerate mTOR inhibitors.
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Therapies that can be discontinued.
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The difficulty in conducting a Phase II–III clinical trial in a rare disease with an existing therapy.
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Future drugs or combination of drugs could perhaps be first tested in patients with known rates of decline in lung function. This would allow each patient in essence to serve as their own control, reducing the numbers needed to enroll.
.
Biomarkers for disease activity are needed that would allow their use as end points, therefore reducing the numbers needed to enroll.
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Expert Rev. Respir. Med. 9(2), (2015)
The next breakthrough in LAM clinical trials may be their design
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1.
El-Chemaly S, Henske EP. Towards personalised therapy for lymphangioleiomyomatosis: lessons from cancer. Eur Respir Rev 2014;23:30-5
13.
Taveira-DaSilva AM, Hathaway O, Stylianou M, Moss J. Changes in lung function and chylous effusions in patients with lymphangioleiomyomatosis treated with sirolimus. Ann Intern Med 2011;154: 797-805. W-292-793
14.
Yao J, Taveira-DaSilva AM, Jones AM, et al. Sustained effects of sirolimus on lung function and cystic lung lesions in lymphangioleiomyomatosis. Am J Respir Crit Care Med 2014;190:1273-82
15.
Yao J, Taveira-DaSilva AM, Jones AM, et al. Sustained effects of sirolimus on lung function and cystic lung lesions in lymphangioleiomyomatosis. Am J Respir Crit Care Med 2014;190(11):1273-82
16.
Cottin V, Harari S, Humbert M, et al. Pulmonary hypertension in lymphangioleiomyomatosis: characteristics in 20 patients. Eur Respir J 2012;40:630-40
17.
Taveira-DaSilva AM, Hathaway OM, Sachdev V, et al. Pulmonary artery pressure in lymphangioleiomyomatosis: an echocardiographic study. Chest 2007;132:1573-8
18.
McCormack FX, Inoue Y, Moss J, et al. Efficacy and safety of sirolimus in lymphangioleiomyomatosis. N Engl J Med 2011;364:1595-606 Seyama K, Kumasaka T, Souma S, et al. Vascular endothelial growth factor-D is increased in serum of patients with lymphangioleiomyomatosis. Lymphat Res Biol 2006;4:143-52
2.
Taveira-DaSilva AM, Moss J. Optimizing treatments for lymphangioleiomyomatosis. Expert Rev Respir Med 2012;6:267-76
3.
Carsillo T, Astrinidis A, Henske EP. Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc Natl Acad Sci USA 2000;97:6085-90
4.
5.
6.
7.
8.
9.
10.
11.
12.
Yu J, Astrinidis A, Henske EP. Chromosome 16 loss of heterozygosity in tuberous sclerosis and sporadic lymphangiomyomatosis. Am J Respir Crit Care Med 2001;164:1537-40 Henske EP, McCormack FX. Lymphangioleiomyomatosis - a wolf in sheep’s clothing. J Clin Invest 2012;122: 3807-16 Goncharova EA, Goncharov DA, Eszterhas A, et al. Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM). J Biol Chem 2002;277:30958-67
24.
Chang WY, Cane JL, Kumaran M, et al. A 2-year randomised placebo-controlled trial of doxycycline for lymphangioleiomyomatosis. Eur Respir J 2014;43:1114-23
25.
Pimenta SP, Baldi BG, Kairalla RA, Carvalho CR. Doxycycline use in patients with lymphangioleiomyomatosis: biomarkers and pulmonary function response. J Bras Pneumol 2013;39:5-15
26.
Taveira-DaSilva AM, Stylianou MP, Hedin CJ, et al. Decline in lung function in patients with lymphangioleiomyomatosis treated with or without progesterone. Chest 2004;126:1867-74
27.
Johnson SR, Whale CI, Hubbard RB, et al. Survival and disease progression in UK patients with lymphangioleiomyomatosis. Thorax 2004;59:800-3
28.
Glassberg MK, Elliot SJ, Fritz J, et al. Activation of the estrogen receptor contributes to the progression of pulmonary lymphangioleiomyomatosis via matrix metalloproteinase-induced cell invasiveness. J Clin Endocrinol Metab 2008;93:1625-33
Zafar MA, McCormack FX, Rahman S, et al. Pulmonary vascular shunts in exercise-intolerant patients with lymphangioleiomyomatosis. Am J Respir Crit Care Med 2013;188:1167-70
29.
Li C, Zhou X, Sun Y, et al. Faslodex inhibits estradiol-induced extracellular matrix dynamics and lung metastasis in a model of lymphangioleiomyomatosis. Am J Respir Cell Mol Biol 2013;49:135-42
19.
Davies DM, de Vries PJ, Johnson SR, et al. Sirolimus therapy for angiomyolipoma in tuberous sclerosis and sporadic lymphangioleiomyomatosis: a phase 2 trial. Clin Cancer Res 2011;17:4071-81
30.
Baldi BG, Ribeiro Carvalho CR. Doxycycline in lymphangioleiomyomatosis: not all questions are answered. Eur Respir J 2014;43:1536-7
31.
20.
Bissler JJ, Kingswood JC, Radzikowska E, et al. Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, double-blind, placebo-controlled trial. Lancet 2013;381:817-24
Johnson SR, Chang WY, Tattersfield AE, et al. Doxycycline in lymphangioleiomyomatosis: not all questions are answered. Eur Respir J 2014;43:1538
32.
Brunelli A, Catalini G, Fianchini A. Pregnancy exacerbating unsuspected mediastinal lymphangioleiomyomatosis and chylothorax. Int J Gynaecol Obstet 1996;52:289-90
33.
Ryu JH, Moss J, Beck GJ, et al. The NHLBI lymphangioleiomyomatosis registry: characteristics of 230 patients at enrollment. Am J Respir Crit Care Med 2006;173: 105-11
34.
Yu JJ, Robb VA, Morrison TA, et al. Estrogen promotes the survival and pulmonary metastasis of tuberin-null cells. Proc Natl Acad Sci USA 2009;106:2635-40
35.
Harari S, Cassandro R, Chiodini I, et al. Effect of a gonadotrophin-releasing hormone analogue on lung function in
Young LR, Vandyke R, Gulleman PM, et al. Serum vascular endothelial growth factor-D prospectively distinguishes lymphangioleiomyomatosis from other diseases. Chest 2010;138:674-81 Young LR, Lee HS, Inoue Y, et al. Serum VEGF-D concentration as a biomarker of lymphangioleiomyomatosis severity and treatment response: a prospective analysis of the Multicenter International Lymphangioleiomyomatosis Efficacy of Sirolimus (MILES) trial. Lancet Respir Med 2013;1:445-52 El-Chemaly S, Goldberg HJ, Glanville AR. Should mammalian target of rapamycin inhibitors be stopped in women with lymphangioleiomyomatosis awaiting lung transplantation? Expert Rev Respir Med 2014;8:657-60 Ando K, Kurihara M, Kataoka H, et al. Efficacy and safety of low-dose sirolimus for
informahealthcare.com
monitoring for MMPs. N Engl J Med 2006;354:2621-2
treatment of lymphangioleiomyomatosis. Respir Investig 2013;51:175-83
References
Review
21.
22.
23.
Mohammadieh AM, Bowler SD, Silverstone EJ, et al. Everolimus treatment of abdominal lymphangioleiomyoma in five women with sporadic lymphangioleiomyomatosis. Med J Aust 2013;199:121-3 A study to determine the effectiveness of escalating doses of RAD001 (Everolimus) in patients with lymphangioleiomyomatosis. Available from: https://clinicaltrials.gov/ct2/ show/NCT01059318 Moses MA, Harper J, Folkman J. Doxycycline treatment for lymphangioleiomyomatosis with urinary
203
Review
El-Chemaly & Henske
Expert Review of Respiratory Medicine Downloaded from informahealthcare.com by Nanyang Technological University on 04/25/15 For personal use only.
lymphangioleiomyomatosis. Chest 2008;133:448-54 36.
Trial of aromatase inhibition in lymphangioleiomyomatosis (TRAIL). Available from: https://clinicaltrials.gov/ct2/ show/NCT01353209
37.
Taveira-DaSilva AM, Steagall WK, Rabel A, et al. Reversible airflow obstruction in lymphangioleiomyomatosis. Chest 2009;136:1596-603
38.
39.
40.
Nebulized or inhaled albuterol for lymphangioleiomyomatosis. Available from: https://clinicaltrials.gov/ct2/show/ NCT01799538 Baldi BG, Albuquerque AL, Pimenta SP, et al. Exercise performance and dynamic hyperinflation in lymphangioleiomyomatosis. Am J Respir Crit Care Med 2012;186:341-8 Baldi BG, de Albuquerque AL, Pimenta SP, et al. A pilot study assessing the effect of bronchodilator on dynamic hyperinflation in LAM. Respir Med 2013;107:1773-80
42.
Puhan MA, Lareau SC. Evidence-based outcomes from pulmonary rehabilitation in the chronic obstructive pulmonary disease patient. Clin Chest Med 2014;35:295-301
44.
45.
46.
47.
Yen KT, Putzke JD, Staats BA, Burger CD. The prevalence of acute response to bronchodilator in pulmonary lymphangioleiomyomatosis. Respirology 2005;10:643-8
41.
43.
https://clinicaltrials.gov/ct2/show/ NCT02009241
Dowman L, Hill CJ, Holland AE. Pulmonary rehabilitation for interstitial lung disease. Cochrane Database Syst Rev 2014;10:CD006322 Rochester CL, Fairburn C, Crouch RH. Pulmonary rehabilitation for respiratory disorders other than chronic obstructive pulmonary disease. Clin Chest Med 2014;35:369-89 Pulmonary Rehabilitation in Lymphangioleiomyomatosis. Available from:
204
Atochina-Vasserman EN, Goncharov DA, Volgina AV, et al. Statins in Lymphangioleiomyomatosis. Simvastatin and atorvastatin induce differential effects on tuberous sclerosis complex 2-null cell growth and signaling. Am J Respir Cell Mol Biol 2013;49:704-9 Goncharova EA, Goncharov DA, Fehrenbach M, et al. Prevention of alveolar destruction and airspace enlargement in a mouse model of pulmonary lymphangioleiomyomatosis (LAM). Sci Transl Med 2012;4:154ra134
48.
Safety of Simvastatin in LAM and TSC (SOS). Available from: https://clinicaltrials. gov/ct2/show/NCT02061397
49.
Parkhitko A, Myachina F, Morrison TA, et al. Tumorigenesis in tuberous sclerosis complex is autophagy and p62/ sequestosome 1 (SQSTM1)-dependent. Proc Natl Acad Sci USA 2011;108:12455-60
50.
Safety study of sirolimus and hydroxychloroquine in women with lymphangioleiomyomatosis (SAIL). Available from: https://clinicaltrials.gov/ct2/show/ NCT01687179
51.
Gottenberg JE, Ravaud P, Puechal X, et al. Effects of hydroxychloroquine on symptomatic improvement in primary Sjogren syndrome: the JOQUER randomized clinical trial. JAMA 2014;312: 249-58
52.
53.
Rosenfeld MR, Ye X, Supko JG, et al. A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy 2014;10:1359-68
lymphangioleiomyomatosis. Cancer Res 2014;74:1996-2005 54.
The tolerability of saracatinib in subjects with lymphangioleiomyomatosis (LAM) (SLAM-1). Available from: https:// clinicaltrials.gov/ct2/show/NCT02116712
55.
McNeish IA, Ledermann JA, Webber L, et al. A randomised, placebo-controlled trial of weekly paclitaxel and saracatinib (AZD0530) in platinum-resistant ovarian, fallopian tube or primary peritoneal cancer dagger. Ann Oncol 2014;25:1988-95
56.
Molina JR, Foster NR, Reungwetwattana T, et al. A phase II trial of the Src-kinase inhibitor saracatinib after four cycles of chemotherapy for patients with extensive stage small cell lung cancer: NCCTG trial N-0621. Lung Cancer 2014;85:245-50
57.
Seyama K, Kumasaka T, Kurihara M, et al. Lymphangioleiomyomatosis: a disease involving the lymphatic system. Lymphat Res Biol 2010;8:21-31
58.
Cui Y, Osorio JC, Risquez C, et al. Transforming growth factor-beta1 downregulates vascular endothelial growth factor-D expression in human lung fibroblasts via the Jun NH2-terminal kinase signaling pathway. Mol Med 2014;20:120-34
59.
El-Chemaly S, Pacheco-Rodriguez G, Malide D, et al. Nuclear localization of vascular endothelial growth factor-D and regulation of c-Myc-dependent transcripts in human lung fibroblasts. Am J Respir Cell Mol Biol 2014;51:34-42
60.
Pimenta SP, Baldi BG, Acencio MM, et al. Doxycycline use in patients with lymphangioleiomyomatosis: safety and efficacy in metalloproteinase blockade. J Bras Pneumol 2011;37:424-30
Tyryshkin A, Bhattacharya A, Eissa NT. SRC kinase is a novel therapeutic target in
Expert Rev. Respir. Med. 9(2), (2015)
