Participants
Patients with mild or moderate aPAP, aged between 18 and 80 years old, were enrolled at two hospitals, including Peking Union Medical College Hospital (PUMCH) and The Affiliated Drum Tower Hospital of Nanjing University Medical School in China.
The inclusion criteria included: (1) patients with a clinical diagnosis of PAP by high-resolution computed tomography (HRCT), further pathologically confirmed by testing for amorphous periodic Acid-Schiff (PAS)-positive granules; (2) a positive serum GM-CSF antibody test which indicated an elevated serum GM-CSF antibody level; and (3) patients eligible for the trial should have progressive or unremitting PAP, defined as worsening or unchanging PaO2 or A–aDO2 over a 3-month period of observation. PAS-positive granules were found either in milky broncho-alveolar lavage fluid (BALF) or in alveolar structures of lung biopsy tissues which were obtained as follows: cytological findings of bronchial lavage fluid only (BAL) (n=13), transbronchial lung biopsy only (TBLB) (n=7), both TBLB and BAL (n=12), percutaneous lung puncture biopsy (n=3), and surgical lung biopsy only (n=1). The GM-CSF antibody test was performed according to the method established by Uchida et al [6, 7]. Our hospital set the cutoff point at 2.39g/mL, with measurements in excess of this value resulting a positive report [8].
Disease severity was assessed with a disease severity score (DSS), with patients with a DSS of 1 to 3 inclusive being included in our study. DSS scores were defined by Inoue et al. as follows [9]: Grade 1: No symptoms and an arterial oxygen partial pressure (PaO2) ³ 70mmHg; Grade 2: PaO2³ 70mmHg with symptoms; Grade 3: PaO2 between 60-70mmHg; Grade 4: PaO2 between 50-60mmHg; and Grade 5: PaO2 below 50mmHg.
Individuals were excluded if they met the following criteria: (1) patients had already received previous GM-CSF therapy or other forms of cytokine therapy, or had undergone lung lavage therapy within the 3 months prior to enrollment; (2) Individuals with PAP resulting from other conditions (e.g. occupational exposure to silica, underlying human immunodeficiency virus infection, respiratory infections, myeloproliferative disorder or leukemia); (3) Individuals with histories of severe allergic or anaphylactic reactions to humanized or murine monoclonal antibodies; (4) Individuals with chronic lung diseases or any other serious medical conditions, or (5) Women who were pregnant, lactating or planned to become pregnant during the study period.
Study Design
This was a multicenter, randomized, open-label clinical trial (clinical trial number: NCT02243228, Inhalation of granulocyte-macrophage colony stimulating factor for autoimmune pulmonary alveolar proteinosis) comprising three sequential periods: high-dose therapy for 3 months, low-dose therapy for 3 months 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 (Figure 1). Patients’ safety questionnaires were reviewed by telephone at 1, 15 and 21 months. Before the therapeutic trial, all participants entered an initial 3-month observation period, during which disease severity and progression were evaluated. Participants that had their PaO2 increase by 10 mmHg or more, or alveolar-arterial oxygen gradient (A–aDO2) decrease by 10 mmHg or more were regarded as having undergone spontaneous improvement and were excluded from enrollment. It should be noted that if a participant was acquainted to the principal investigator as a patient with a well-documented history showing an unremitting aPAP state, he/she could be enrolled into the study without this observation period. After 3-months of observation, all unremitting PAP patients underwent a stratified randomization based on their DSS at the time of enrollment to ensure equal representation of patients with various disease severities in both the treatment group and the placebo group using a random number table. The randomization was blinded to both the patients and the investigators.
Recombinant human GM-CSF (rhGM-CSF) was administered to patients in treatment group by inhalation as previously described [10]. 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) [11]. Treatment was designed according to a previous study[12], 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 were not prescribed any kind of treatment related to PAP (including GM-CSF, WLL or anti-CD20, etc.) but had the same follow up plan as the treatment group.
In previous published studies, patients inhaling GM-CSF had a mean change in A–aDO2 of 11 mmHg [12]. Thus, the target sample size was 25, chosen to give a detection power of 90%, allowing for a 5% incidence of type-I error. After taking other outcome measurements and participant dropout into consideration, the study size was increased to 35-45 patients.
The study was approved by the institutional review board of PUMCH (Approval No. S-717) and reviewed by the institutional review boards at the Affiliated Drum Tower Hospital of Nanjing University Medical School. All participants provided informed written consent prior to enrollment.
Assessments
Clinical information including history, symptoms, serological (including lactate dehydrogenase, carcinoembryonic antigen levels) and radiological features, pulmonary function testing results and physical examination results were obtained at each visit during the study. Arterial blood gas analysis (ABG) tests were performed with patients that had been breathing room air for at least 15 minutes. A low dose quantitative computed tomography of the chest (in PUMCH) or high resolution CT (HRCT) of the chest (in The Affiliated Drum Tower Hospital of Nanjing University Medical School) was obtained before and after GM-CSF therapy and evaluated in a blinded fashion by a board-certified radiologist. The original CT measurements were collected, and the total lung volume and mean lung density were automatically calculated and post-processed with Pulmo 3D (syngo. via, version VA 30, Siemens Healthcare, Germany) for the automatic segmentation of the pulmonary parenchyma by excluding the intrapulmonary vessels following the process published by one of our co-authors, Dr. Sui [13].
Intergroup differences in the change of A–aDO2 from baseline to the end of treatment were defined as primary endpoints.
Other data, representing the 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 follows: 1) a 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 PaO2 of more than 10mmHg, or increase in A–aDO2 of more than 10mmHg; or 4) a worsened chest HRCT independently confirmed by two physicians. Monitoring for adverse events was conducted during the study, looking for airway hypersensitivity, fever, mylagia, arrhythmia and potential effects on the circulatory system.
All blood tests were performed in laboratories affiliated with the two hospitals, both of which have China’s quality management certification. Serum levels of GM-CSF antibody were tested at PUMCH.
All of the data was collected and stored in the database system founded by Beijing Yikang Healthcare Technology Co.
Statistical analysis
All statistical analyses were performed using 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 X2 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.