Inhaled granulocyte-macrophage colony stimulating factor for mild-to-moderate autoimmune pulmonary alveolar proteinosis - a phase II randomized study for 6 months and follow-up to 24 months

Background: Treatment of autoimmune pulmonary alveolar proteinosis (aPAP) by inhaled granulocyte-macrophage colony stimulating factor (GM-CSF) is considered safe and effective. Evidences of benefit from GM-CSG inhalation for mild to moderate aPAP patients are limited. Methods: In this multicenter, randomized, open-labeled clinical trial, 36 aPAP patients with mild to moderate disease severity were randomized into either GM-CSF treatment group or control group. Inhaled GM-CSF was prescribed for 6 months, and patients were followed-up for another 18 months without treatment. Physiological features of the patients were analyzed. Results: There were 36 patients (19 in treatment group, 17 in control group) included. No significant difference in primary endpoints measured by the change of alveolar arterial oxygen gradient (A-aDO2) from the baseline value to the values obtained during treatment or during the following 18-month non-treatment observation period [control group vs. treatment group: 0.51±12.09 mmHg vs. -0.35±13.76 mmHg, p=0.848 (3 month); 1.85±11.21 mmHg vs. 7.31±8.81 mmHg, p=0.146 (6 months); 6.05±11.14 mmHg vs. 6.61±10.64mmHg, p=0.899 (24 months)]). Percentage of diffusion capacity predicted (DLCO%) and percentage of total lung capacity predicted (TLC%), however, were significantly improved in the treatment group at the end of the study (P=0.010 and 0.027). St. George Respiratory questionnaire (SGRQ) scores were better after 6 months treatment with GM-CSF than control group, and the benefits of treatment were maintained throughout the observation period. No severe side effects were observed during the study. Conclusion: Six months of inhaled GM-CSF treatment had no effect on the alveolar–arterial oxygen gradient in patients with mild to moderate pulmonary alveolar proteinosis. There were changes in some clinical or laboratory measures, but no clinically important changes were noted at the end of study.

gradient in patients with mild to moderate pulmonary alveolar proteinosis. There were changes in some clinical or laboratory measures, but no clinically important changes were noted at the end of study.

Background
Autoimmune pulmonary alveolar proteinosis (aPAP, previously known as idiopathic PAP) is a rare interstitial lung disease elicited by the formation of autoantibodies which neutralize the activity of granulocyte-macrophage colony stimulating factor (GM-CSF), consequently decreasing macrophage clearance of surfactant [1]. Currently, the standard treatment strategy for PAP is whole lung lavage (WLL). About 70% patients need another WLL within 3 years due to recurrence [2,3]. Patients who undergo WLL require general anesthesia and double-lumen endotracheal intubation, which means only hospitals with experienced physicians can perform the procedure. Considering the recurrence rate and the cumbersome procedure of WLL, whether or not patients with mild or moderate disease should obtain WLL is a matter of controversy.
Inhaled GM-CSF therapy has become an alternative option for aPAP patients not only due to its effectiveness and safety [4,5], but also because it is a convenient treatment method for patients who are reluctant to receive WLL. Previous studies included small sample sizes, and as a result, disease severity has not been stratified. Nevertheless, whether patients with mild or moderate disease will benefit from the GM-CSF treatment over the long term is still unclear.
We prospectively evaluated if inhaled GM-CSF would delay disease progression in patients with mildto-moderate aPAP over a two-year period. We designed a 6-month treatment and 18-month follow-up observation.

Baseline demographic information
Forty-two aPAP patients were screened and 36 patients were randomized (19 in the treatment group and 17 in the control group). After 24 months of follow up, 26 patients (72.2%, 15 from the treatment group and 11 from the control group) completed the study. The period of recruitment and follow up was from July 20, 2014 to July 6, 2018 after the last enrolled patient completed his 24 months follow up. In the treatment group, one patient deteriorated at 3 months and required rescue therapy (WLL).
Another patient lost follow up at 1 month and two more patients withdrew at 6 months. In the control group, 4 patients deteriorated at 3 months and required rescue therapy (one received GM-CSF inhalation, two received WLL and one was prescribed traditional medicine). 2 patients withdrew at 21 months. (Fig. 1) In 36 patients, the most common presenting symptom was dyspnea (20/36, 55.6%), followed by cough (13/36, 36.1%) and phlegm (6/36, 16.7%) and chest pain (3/36, 8.3%). 4 out of our 36 patients 6 months (inter-quartile range is from 0 to 60 months) in our patients. All of our patients had extent bilateral pulmonary infiltrates confirmed by HRCT.
Demographic information of the 36 patients entered the study is shown in Table 1. There were no significant differences in demographic information between the two groups including age and sex. No significant differences were found in patients' disease severity markers at baseline, including symptoms, ABG, pulmonary function tests, 6 minutes walking distance (6MWD) and anti-GM-CSF antibody levels between the treatment group and the control group.  Fig. 2A). The change of PaO 2 level from baseline to 3 and 6 months treatment, and during the following 18 months also showed no significant difference between the two groups (Fig. 2B). The actual level of A-aDO 2 and PaO 2 showed no differences during both the treatment period and follow up period as well ( Fig. 2C and D) (Tables 2 and 3). Table 2 The clinical parameter of the effects of inhaled GM-CSF during the 6 months treatment periods.  The diffusion capacity and total lung capacity were improved by the end of study The DLCO% and TLC% showed significant differences between the treatment group and the control group by the end of the study ( Fig. 3A  The SGRQ scores increased after 3 months and 6 months of inhaled GM-CSF treatment and 18 months follow-up Meanwhile, we can find obvious difference in patients' quality of life between the treatment group and control group, as measured by SGRQ. The total SGRQ score was improved after 6 months of GM-CSF treatment compared to the no treatment group, and the benefits were nearly continuously maintained throughout the 18-month observation period. Similar trends can be observed in symptom score, activity score and effect score, but not all time points show significant differences between the two groups. (Fig. 4) Quantitative CT did not find difference after treatment There was no significant difference in the total lung volume and mean lung density between the treatment group and control group. (Table 2 and supplemental Table 1) Time to rescue therapy during the 24-month study was not improved There was no significant difference in time to rescue therapy between the treatment group and control group. Kaplan-Meier Curve analysis for the two groups was shown in  (Table 4) Table 4 The side-effect of patients with aPAP during the GM-CSF treatment and follow up period. No other significant safety and tolerability differences were observed between the two groups during the study.
Other details of side effects happened during the study can be found in the supplementary.

Discussion
In the present study, we prospectively evaluated the effects of inhaled GM-CSF on mild-to-moderate autoimmune pulmonary alveolar proteinosis (aPAP) patients. In contrast to previous report, no obvious effects were found in our study. During the 6 months treatment and 18 month subsequent observation, the primary endpoint, A-aDO 2 did not changed. Health-related quality of life as measured as SGRQ improved from 3 months of treatment and maintained to 24 months. Marginal improvement was also noted that TLC and DLCO were improved at the end of the study. This research provides valuable clinical data and experience for inhaled GM-CSF treatment in aPAP patients who do not meet the criteria for WLL.
Current therapy for PAP patients involves the physical removal of surfactant using a procedure in which the lungs are repeatedly filled with saline and emptied-WLL-which is invasive, inefficient, and is not widely available. Some authors reported that fever, hypoxemia, fluid leakage and other complications occurred in patients treated with WLL [6]. Meanwhile, the media time to next WLL is around 15 months [7], and about 30%-57.6% of patients requiring further therapy after the first WLL [8,9]. Though no consensus of the indication for WLL in treating PAP, most physicians believe that patients with PaO 2 of less than 70 mmHg on room air or an alveolar-arterial [A-a] oxygen gradient of more than 40 mmHg, or patients with disease progression should receive WLL as treatment [6]. In a cohort study from our center, 33% patients are stable or experience spontaneous remission [9], and the spontaneous remission rate varies from 8-18% in different reports [7,[9][10][11]. Considering the rate of spontaneous remission, rate of recurrence and the cumbersome procedure of WLL, it becomes a critical question whether GM-CSF inhalation could become a primary treatment for mild to moderate aPAP patients.
After GM-CSF was confirmed to play an important role in the disease mechanism of aPAP, the efficacy of exogenous GM-CSF replacement was assessed in previous report. The response rate to this treatment varied and the efficacy rate was 62-100% when using inhaled GM-CSF [5,11,12] while the efficacy rate was 43-75% when using subcutaneously administered GM-CSF [13,14]. Because of better responsiveness and tolerance, the use of inhaled GM-CSF is generally recommended [4] .
In previous studies, inhaled GM-CSF treatment was prescribed in patients with moderate to severe disease [15][16][17][18][19], and the mean PaO 2 level in a large prospective study of inhaled GM-CSF treatment on aPAP patients is 61.7 ± 1.4 mmHg [11]. During preparation of our manuscript, a randomized placebo-controlled study of inhaled GM-CSF was published, A-aDO 2 and CT density quantitative measurement were significantly improved though they concluded that clinical benefits were not significant [20]. The major differences in design between our study and Tazawa  However, Tazawa, et al has answered this question with a randomized placebo-controlled study. We believe GM-CSF could be beneficial for those with PaO 2 less than 70 mmHg.
We found that inhaled GM-CSF therapy is a well-tolerated choice for aPAP patients as previous studies showed [11,[15][16][17]. Though more than half of our patients in the GM-CSF group were found have slight increases in amino-transferase levels, and a number of abnormal liver function results were observed in the GM-CSF treatment group, the elevation of transaminase levels were all slight and no medical intervention was needed for all patients. All patients remained stable or gradually improved after the cessation of alcohol and stopping intake of possible related combination medicines.
Therefore, inhaled GM-CSF therapy is a safe and convenient choice for patients.
Our research has some limitations. Firstly, the sample size of the study was small, and there was no enough DSS3 patients for analysis. Our estimated target sample size was based on the prior results of patients population with more disease severity [11], which may underestimate the sample size actually needed. Secondly, more patients from the control group dropped out of the study during the observation period, which might affect the evaluation of effectiveness for comparing these two groups. Thirdly, the patients in our study did not receive a tailored dosage of GM-CSF treatment, nor did they receive prolonged therapy after the 6 months of treatment, which may make some latent responders, requiring higher dosages or longer treatment time for a positive response, remain hidden.

Conclusions
Six months of inhaled GM-CSF treatment had no effects on the alveolar-arterial oxygen gradient in patients with mild to moderate pulmonary alveolar proteinosis. At the dose we used, there were changes in some clinical or laboratory measures, but no clinically important changes were noted at the end of study. Our study is an important complement for efficacy in aPAP patients with mild to moderate disease severity.

Methods Participants
Patients with mild or moderate aPAP, aged between 18 and 80, were enrolled at two hospitals, was followed the method Uchida et al established [21,22] and revised the cutoff point (above 2.39 g/ml) performed by our hospital [23].
Disease severity was assessed with a disease severity score (DSS), with patients having a DSS between 1 and 3 being included in our study. DSS scores were defined based on a previous study as follows [24]: Recombinant human GM-CSF (rhGM-CSF) was administered to patients in treatment group by inhalation as previously described [15]. 150 µg of rhGM-CSF was dissolved in 2 ml of sterile saline, and was inhaled as an aqueous aerosol using an LC-PLUS nebulizer with a manual interrupter valve connected to a PARI Turbo BOY compressor (PARI GmbH, Starnberg, Germany) [25]. The drug was donated by North China Pharmaceutical Corporation (NCPC) and nebulizers were bought from PARI.
Treatment was designed according to a previous study [11], including 3 months of high-dose GM-CSF administration (150 µg twice daily every other week) and another 3 months of low-dose administration (150 µg once daily every other week), serving as induction and maintenance therapy, respectively.
In the control group, patients did not prescribe any kind of treatment related to PAP (including GM-CSF, WLL or anti-CD20, et al) but have the same follow up plan as treatment group.
In previous published studies, patients inhaling GM-CSF had a mean change in A-aDO 2 of 11 mmHg [11]. Thus, the target sample size was 25, chosen to give a detection power of 90%, allowing for a 5% incidence of type-I error. Considering other outcome measurements and participant dropout, the study size was increased to 35-45 patients. Germany) for the automatic segmentation of the pulmonary parenchyma by excluding the intrapulmonary vessels following the process published by one of our co-author, Dr. Sui [26].
Intergroup differences in the change of A-aDO 2 from baseline to the end of treatment were defined as primary endpoints.
Other data, representing efficacy of GM-CSF inhalation, were also evaluated as secondary endpoints, including pulmonary function test differences between the treatment group and the control group (forced vital capacity [FVC], total lung capacity [TLC], diffusing capacity for carbon monoxide [DLCO] or diffusing capacity for carbon monoxide corrected for alveolar volume [DLCO/VA]), 6 minutes walking distance differences between the groups, and relapse time in the two groups. The definition of relapse was as follow: 1) new requirement for whole lung lavage (WLL) or any other kind of treatment (including traditional medicine, subcutaneous injection or GM-CSF inhalation) due to disease progression; or 2) PAP death; or 3) reduction in PaO 2 of more than 10 mmHg, or increase in A-aDO 2 of more than 10 mmHg; or 4) a worsened chest HRCT independently confirmed by two physicians. Adverse events were monitored during the study, including airway hypersensitivity, fever, mylagia, arrhythmia and potential effects on the circulatory system.
All blood tests were performed in the laboratories affiliated with the two hospitals, both of which have the quality management certification of China. Serum levels of GM-CSF antibody were tested in PUMCH.
All the data were collected and stored in the database system founded by Beijing Yikang Healthcare Technology Co.

Statistical analysis
All statistical analyses were performed using a personal computer and SPSS 20.0 software. Numeric results were presented as either the mean ± SD or the median and inter-quartile range. Metric variables were shown as the mean and categorical variables were given in terms of frequencies and percentages. The X 2 test was used to analyze proportions of variables. For group comparisons, the unpaired t tests and Wilcoxon rank-sum test were used to evaluate the differences in normally distributed variables. Kaplan-Meier Curve analysis was used to analyze time for relapse in the two groups. All P values reported were two-sided.    The DLCO% and TLC% showed significant differences between the treatment group compared with the control group at the end of the study (P<0.05, respectively, A and B).

List Of Abbreviations
However, other pulmonary function tests, including FVC and DLCO/VA, did not show any significant differences between the treatment group and the control group, both during the 6-month treatment period and the 18-month follow-up period (C and D  The SGRQ score during the 24-month study period. A, total SGRQ score; B, SGRQ symptom score; C, SGRQ activity score; D, SGRQ effect score. *:P <0.05, **: P <0.01.

Figure 5
No disease progression rate (measured by time to rescue therapy) during the 24 months study has no significant difference between the treatment and control groups. P >0.05.

Figure 6
The clinical trial comprising three sequential periods: high-dose therapy for 3 months (150 μg twice daily every other week), low-dose therapy for 3 months (150 μg once daily every other week) and observation for 18 months. Study visits during treatment were designed at 0, 1, 3 and 6 months. Thereafter, patients were followed up by visits at 9, 12, 15, 18, 21 and 24 months.