Long-term results of sirolimus treatment in lymphangioleiomyomatosis: a single referral centre experience

There are few published data on long-term treatment with sirolimus in lymphangioleiomyomatosis (LAM). The objective of this study was to describe the long-term effect of sirolimus in a series of LAM patients followed up in a referral centre, focusing on pulmonary function. We retrospectively reviewed a series of 48 patients with LAM diagnosed, followed up and treated with sirolimus in a single centre. Response to sirolimus was evaluated at 1 and 5 years. A negative sirolimus response was defined as an FEV1 decline greater than − 75 ml/year. A mixed-effects model was used to estimate the longitudinal changes in FEV1 (average slope), both as absolute (ml/year) and as predicted values (%predicted/year). From a total of 48 patients, 9 patients underwent lung transplantation and 4 died during the study. Mean (95% CI) FEV1 slope over 5 years was − 0.14 (− 26.13 to 25.85) ml/year in the whole LAM group, 42.55 (14.87 to 70.22) ml/year in the responder group, − 54.00 (− 71.60 to − 36.39) ml/year in the partial responder group and − 84.19 (− 113.5 to − 54.0) ml/year in the non-responder group. After 5 years of sirolimus treatment 59% had a positive response, 30% had a partial response and 11% had a negative response. Our study found that sirolimus treatment had a positive long-term effect on most LAM patients.

www.nature.com/scientificreports/ Sirolimus was introduced, as an off-label use, in LAM patients in November 2007 following the publication of the first trial in renal angiomyolipomas 10 . The objective of the present study was to review the long-term effect of sirolimus in a series of LAM patients followed up in a referral centre, focusing on pulmonary function.

Patients and clinical assesment
The study retrospectively included all patients with LAM diagnosed, treated with sirolimus and followed up in a tertiary referral centre from January 1990 to January 2020. Data compiled included clinical findings, diagnostic methods, treatment, lung function, presence of tuberous sclerosis and extrapulmonary involvement such as renal angiomyolipoma, pleural or abdominal complications. The Ethics Committee of the Vall d'Hebron University Hospital (Barcelona, Spain BSG-SIR-2019-01) approved the study. Informed consent was waived due to retrospective nature of the study by The Ethics Committee of the Vall d'Hebron University Hospital. All methods in this study were performed in accordance with the relevant guidelines and regulations. Pulmonary function tests were performed according to European Respiratory Society guidelines 11 . All FEV 1 values in our study were obtain without bronchodilators. Patients with LAM underwent spirometric variables and diffusing capacity -at baseline, 1 year, 2 years, 3 years, 4 years and 5 years. Thoracic and abdominal CT scan were performed in all patients. Lung biopsy was performed only in patients with exclusive pulmonary involvement; biopsy was generally avoided if patients had lung cysts associated with renal angiomyolipomas.
Time to event was calculated as the number of years between sirolimus initiation to the date of lung transplantation or date of death, whichever came first.
Patients were classified into two cohorts according to the availability of sirolimus at diagnosis: a historical sirolimus cohort (HSC) of patients for whom sirolimus was not available at diagnosis but who received the drug after 2007 and a contemporary cohort (CC), in whom sirolimus was started immediately after the diagnosis.
Sirolimus treatment. From November 2007, sirolimus was indicated in the case of FEV 1 < 80%, angiomyolipoma bigger than 4 cm of diameter or multiple angiomyolipomas. Sirolimus was prescribed at a dose between 1 and 4 mg once daily adjusted to obtain target trough blood levels between 5 and 15 ng/ml measured with liquid chromatography-mass spectrometry. Adverse effects and drug interactions were also recorded.
We defined the sirolimus response based on the rate of FEV 1 decline in LAM patients without specific treatment, which has been reported as − 75 ml/year 12 . Age-related physiological decline in healthy adults under 60 years old has been reported to be around − 20 ml/year [13][14][15] . Consequently, improvement, stabilization or loss of FEV 1 similar to the physiological decline was considered a positive response to sirolimus. Patients who had an FEV 1 decline rate between − 20 and − 75 ml/year were considered partial responders. Non-responders had an FEV 1 decline greater than − 75 ml/year. Sirolimus response was assessed at 1 year and long-term response at 5 years. Patients who underwent LT before completing 1 or 5 years of sirolimus treatment were not included in the respective analysis.
Angiomyolipomas' shrinkage rate was calculated as the change in maximum diameter after 1 year of sirolimus treatment. Patients with a decrease or stabilization in angiomyolipoma size were considered responders.

Statistical analysis.
Mixed-effect models were used to estimate the longitudinal changes in FEV 1 (average slope), both as absolute (ml/year) and as predicted values (%predicted/year). In these analyses, time was entered as a random effect. We then categorised individuals into three groups: (1) responders (if the individualised estimate was less than − 20 ml/year), (2) partial responders (if the individualised estimate was between − 20 and − 75 ml/year); and (3) non-responders (if the individualised estimate was greater than − 75 ml/year). Qualitative data are expressed as frequencies and percentages. Normally-distributed quantitative data are expressed as mean ± SD; non-normally-distributed data are expressed as median and interquartile range (IQR). The demographic and clinical characteristics of the HSC and CC were compared using ANOVA or chi-square test, as appropriate. Pulmonary function tests (PFT) of non-responders, partial responders and responders to sirolimus were compared using the Mann Whitney test. Time from sirolimus initiation to death/transplantation was assessed using Kaplan-Meier curves. Cox regression analysis was used to compared HSC and CC. The 95% CI was obtained from the mean estimates. A p-value < 0.05 was considered statistically significant. Data were analysed using STATA version 14 (StataCorp, College Station, TX, USA) and SPSS version 27 (IBM Corp., Armonk, NY, USA).

Results
Forty-eight LAM patients diagnosed and followed-up in our outpatient clinic and treated with sirolimus were recruited: 19 (40%) patients correspond to HSC and 29 (60%) to CC. Four (8.3%) of them had LAM associated with TSC. The median age at onset of symptoms and at diagnosis was 36 (30-44) and 38 (34-46) years, respectively. Thirty-nine (81%) patients were pre-menopausal at diagnosis and 9 (19%) were post-menopausal. Three (6%) patients were smokers at diagnosis and 14 (29%) were ex-smokers. Dyspnoea was the most common presenting symptom in 17 (35%) patients followed by pneumothorax in 15 (31%) patients. Mean FEV 1 at diagnosis was 68 ± 26% of the predicted. On thoracic CT, all patients had multiple cystic images with bilateral distribution, predominantly in the middle and lower lobes. The method of diagnosis was surgical lung biopsy in 28 (58%) patients, clinicoradiological findings in 12 (25%) and transbronchial cryobiopsy in 8 (17%). Three (6%) diagnoses were incidental in asymptomatic patients.
During the study period, 4 (8%) patients died, only one of them had received LT and died from an acute cellular rejection. The other causes of death were respiratory failure, renal failure and cholangiocarcinoma. A comparison between the HSC and CC is presented in Table 1.
Survival rates free from death and/or transplantation at 1-and 5-years after sirolimus initiation were 87% and 73% respectively (Fig. 1).
Safety. Side effects were recorded in 16 (33%) patients and tended to occur shortly after starting treatment and decrease over time. The most common side effect was oral aphthous ulcers in 8 (17%), ovarian cysts in 3 (6%), severe increase in serum cholesterol in 2 (4%), liver enzyme abnormalities in 2 (4%), anasarca in 1 (2%) and leg  www.nature.com/scientificreports/ swelling in 1 (2%). Only 1 patient discontinued sirolimus treatment due to fluid retention and in 3 cases sirolimus was temporarily withdrawn due to oral aphthous ulcers.
Efficacy. At the end of the study, of 48 sirolimus-treated patients, 38 (79%) had had treatment for 1 year and 27 (56%) had had a minimum of 5 years' treatment ( Fig. 3).
After 1 year, a positive response to sirolimus was observed in 23 (60%) patients and a partial response in 11 (29%) (Fig. 3). Long-term efficacy of sirolimus was studied in 27 patients who had received 5 years or more of sirolimus treatment: 59% were responders, 30% were partial responders and 11% non-responders. There were no differences in FVC, FEV 1 or DL CO at diagnosis or before sirolimus treatment between responders, partial responders and non-responders; see Table 2. However, significantly differences were observed in FEV 1 after 5-years of sirolimus treatment having non-responders lower values.
Of note, from the responders group, four patients developed chylothorax and only one patient was treated by pleurodesis before sirolimus was initiated. In the other 3 patients, sirolimus was started due to the chylothorax.
Eleven patients with angiomyolipoma were treated with sirolimus for at least one year. Table 4 shows angiomyolipoma sizes before and after 1 year of sirolimus treatment: 10 (91%) patients showed a response to treatment and only 1 (9%) had an increase in tumour size. During follow-up, no patient presented symptoms related to angiomyolipoma.   Table 3. FEV 1 response to sirolimus in 27 patients treated with sirolimus for 5 years. Data are shown as Mean (95% CI).

Discussion
There are few data on long-term pulmonary function results in LAM patients receiving sirolimus. The main finding from this study was that 59% of sirolimus-treated patients were considered responders because they had a sustained spirometric improvement after 5 years of treatment. The MILES trial found that the FEV 1 slope in the sirolimus group gained 1 ± 2 ml per month during the first year of treatment 8 . Although this difference was statistically significant between sirolimus and placebo groups, a substantial number of patients receiving sirolimus treatment had a decrease in FEV 1 during this period. One recent Japanese series of 63 patients treated with sirolimus for 2 years showed an FEV 1 decline rate of between 0.67 and 2.09 ml per month but did not differentiate between responders and non-responders 16 . One study that reported long-term effects of sirolimus in 12 patients found differences in FEV 1 before and after treatment of − 55 ± 7 ml and − 5 ± 12 ml per year, respectively, with a follow-up time of 4.6 years on sirolimus 9 . A Chinese study which evaluated long-term sirolimus effect in 32 patients also found stabilization of pulmonary function after 4 years of sirolimus treatment 17 . It is clear that some patients treated with sirolimus do not respond to treatment and continue to lose lung function 18 . In the present series, 59% of sirolimus-treated patients were considered responders because had a mean FEV 1 slope over 5 years of 42 ml/year vs a mean FEV 1 slope of − 54 ml/year in the partial responders vs − 84 ml/year in the non-responders. We have also assessed the change of FEV 1 % predictive increasing in mean 2.17% in responders and dropping 2.96% in non-responders per year. In summary, of those treated for 5-years sirolimus treatment led to a clear improvement in lung function in half of the patients and had a modest effect in another 30%. Only 11% of sirolimus-treated patients had no improvement in FEV 1. Another relevant aspect is the potential role of sirolimus as a preventive treatment of pleural complications. The present study supports the idea that sirolimus may have some preventive effect against pneumothorax and, probably, chylous effusions. Intuitively, it could be that chylothorax disappearances after sirolimus treatment might be the cause of pulmonary function improvement in those patients with chylothorax. But, chylothorax improvement is in fact secondary to sirolimus treatment. This good response of patients with chylothorax to sirolimus treatment had also been observed by Takada and colleagues 16 .
In the present series, only 9 patients treated with sirolimus were submitted to LT. The role of LT for LAM patients has evolved over time. During the 1980s, 1990s and early 2000s, the only choice for LAM patients with respiratory failure was LT, and LAM was a rare indication for LT 19 . In our series with more than 1100 LTs, LAM represents 2.5% of the total LT caseload. There was only one LAM patient who received LT and belong to CC.
The differences between the HSC and the CC, could be explained by several factors. Although the introduction of sirolimus in the treatment of LAM seems the likely main cause of these differences, other factors such as increased knowledge and interest in LAM in recent years, the development of a specialist referral centre, and earlier diagnosis with higher functional reserve could explain the better results in the CC cohort. All these factors are likely to have influenced the overall improved survival in LAM patients in the last decade and the current perception that LT in LAM will become anecdotal. Perhaps, though, this scenario is somewhat optimistic: we must add a note of caution as our data showed that sirolimus had no effect in 11% of patients with LAM at 5-years.
We observed a positive effect in most cases of angiomyolipoma after one year of treatment, but no effect in 9%. These results are in line with the study published by Bissler et al. that reported no effect in 20% of cases 10 . Also, we found that more than half of the patients who had a positive response in terms of reduction and stabilization of angiomyolipomas had a positive pulmonary functional response. Bissler and colleagues reported the same effect in 7 out of 11 patients who had PFT data at 1 year of sirolimus treatment. Davies et al. also reported, in 12 patients, a 50% decrease in angiomyolipoma size without a significant improvement in lung function during sirolimus therapy. None of the authors reported any data on a correlation between angiomyolipoma and pulmonary function response 10,20 . www.nature.com/scientificreports/ Regarding safety, we recorded side effects in 33% of patients, mainly mild or moderate, and only 1 patient discontinued sirolimus due to side effects. Other authors observed the same spectrum of side effects but in 50% to 59% of patients 16,21 . We did not observe any cases of pneumonitis, something that has previously been described 21 . One of the reasons for these differences could be the retrospective design of our study in contrast with other prospective studies 16,21 . However, we consider that all serious adverse events were reported and recorded.
The main limitation of the study is its retrospective, single-centre design. The number of LAM patients included in the study was conditioned by the rarity of the disease and covers an extended period of 30 years; this is a limitation for most LAM studies. The strengths include the homogeneity of the clinical care and criteria, as most patients were treated by the same team, who had extensive experience with mTOR inhibitors, LT, and the long-term follow-up of sirolimus-treated patients.
In conclusion, sirolimus treatment in LAM has a long-term positive or moderate effect in most patients. LT remains the treatment of choice in LAM patients with respiratory failure. Determining the long-term effects of sirolimus on survival, the mechanisms that preclude treatment response and identifying potential biomarkers are important future priorities.