REDACTED PROTOCOL - The Lancet



Protocol of the studyA multicenter prospective cohort study to compare the efficacy and safety of prednisone and azithromycin combined with or without MSCs in bronchiolitis obliterans syndrome after allo-HSCTApplicant Institution: Nanfang Hospital, Southern Medical UniversityPrincipal institution: Nanfang Hospital, Southern Medical University Version:2.0Date: 20140806Remark: The primary version of this protocol was in Chinese. We have translated it into English.Table of Contents TOC \o "1-3" \h \z \u List Of Abbreviations PAGEREF _Toc6601806 \h 4Protocol Summary PAGEREF _Toc6601807 \h 51. Background PAGEREF _Toc6601808 \h 72. Objectives PAGEREF _Toc6601809 \h 82.1 Primary Objective PAGEREF _Toc6601810 \h 82.2 Secondary Objective PAGEREF _Toc6601811 \h 83. Study Design PAGEREF _Toc6601812 \h 84. Subject Selection Criteria PAGEREF _Toc6601813 \h 94.1 Subject Selection Criteria PAGEREF _Toc6601814 \h 94.1.1 Number of Subjects PAGEREF _Toc6601815 \h 94.1.2 Inclusion Criteria PAGEREF _Toc6601816 \h 94.1.3 Exclusion Criteria PAGEREF _Toc6601817 \h 104.2. Removal Criteria PAGEREF _Toc6601818 \h 104.3. Withdrawal Criteria PAGEREF _Toc6601819 \h 105. Study Procedures PAGEREF _Toc6601820 \h 115.1 Treatment Selection PAGEREF _Toc6601821 \h 115.2 Study Treatment PAGEREF _Toc6601822 \h 115.2.1 MSC Group PAGEREF _Toc6601823 \h 115.2.2 Non-MSC Group PAGEREF _Toc6601824 \h 126. Efficacy Assessments PAGEREF _Toc6601825 \h 126.1 Definition PAGEREF _Toc6601826 \h 126.2. Primary Endpoint PAGEREF _Toc6601827 \h 136.3 Secondary Endpoints PAGEREF _Toc6601828 \h 136.4 Schedule and Methods of Endpoints Assessments PAGEREF _Toc6601829 \h 137. Safety PAGEREF _Toc6601830 \h 147.1 Safety Endpoints PAGEREF _Toc6601831 \h 147.1.1 Vital signs PAGEREF _Toc6601832 \h 147.1.2 Clinical symptoms PAGEREF _Toc6601833 \h 147.1.3 Laboratory abnormalities: PAGEREF _Toc6601834 \h 147.2 Toxicity Assessment Of Adverse Events (AE) and Serious Adverse Events (SAE) PAGEREF _Toc6601835 \h 158.Adverse Events PAGEREF _Toc6601836 \h 158.1 Definition of an AE PAGEREF _Toc6601837 \h 158.2 Relationship of AEs PAGEREF _Toc6601838 \h 168.3 Serious Adverse Events PAGEREF _Toc6601839 \h 168.3.1 Definition PAGEREF _Toc6601840 \h 168.3.1 Reporting of Serious Adverse Events PAGEREF _Toc6601841 \h 179. Rules of Withdrawal PAGEREF _Toc6601842 \h 179.1 Subjects withdraw from the study PAGEREF _Toc6601843 \h 179.2 Premature Termination of the Study PAGEREF _Toc6601844 \h 1810. Rules of Follow-Up PAGEREF _Toc6601845 \h 1810.1 Follow-up Period PAGEREF _Toc6601846 \h 1810.2 Visit Scheduling PAGEREF _Toc6601847 \h 1810.3 Contents PAGEREF _Toc6601848 \h 1811. Data Analysis and Statistical Considerations PAGEREF _Toc6601849 \h 1911.1 Hypotheses PAGEREF _Toc6601850 \h 1911.2 Study Design Considerations PAGEREF _Toc6601851 \h 1911.2.1 Sample Size Assumptions PAGEREF _Toc6601852 \h 1911.2.2 Primary Efficacy Endpoint PAGEREF _Toc6601853 \h 2011.2.3 Secondary Efficacy Endpoints PAGEREF _Toc6601854 \h 2011.3 Data Analysis Considerations PAGEREF _Toc6601855 \h 2011.3.1 Baseline Data PAGEREF _Toc6601856 \h 2011.3.2 Analysis of efficacy PAGEREF _Toc6601857 \h 2011.3.3 Analysis of safety PAGEREF _Toc6601858 \h 2112. Materials for the Study PAGEREF _Toc6601859 \h 2113. Ethical Considerations PAGEREF _Toc6601860 \h 2113.1 Responsibility Of Investigators PAGEREF _Toc6601861 \h 2113.2 Informed Consent Process PAGEREF _Toc6601862 \h 2213.3 Protection of Subjects’ Personal Data PAGEREF _Toc6601863 \h 2214. Administrative Requirements PAGEREF _Toc6601864 \h 2215. Quality Control Of Data (the same as the primary study) PAGEREF _Toc6601865 \h 22List Of Abbreviationsallo-HSCTAllogeneic hematopoietic stem cell transplantationAEsAdverse eventsBOSBronchiolitis obliterans syndromecGVHDChronic graft-versus host diseaseCRF Case Report FormFEV1Forced expiratory volume in 1 s LFSLeukemia-free survivalMSCsMesenchymal stem cellsOSOverall survivalPFTPulmonary function testProtocol SummaryApplicantNanfang Hospital, Southern Medical UniversityPhasePhase I/IIIndicationAllo-HSCT recipients with BOS diagnosed less than 6 months.ObjectivesTo compare the efficacy and safety of prednisone and azithromycin combined with or without MSCs in BOS after allo-HSCTStudy designphase I/II, multicenter, open-label, prospective cohort studyRequired subject60-62 subjectsStudy sites6 study sites (Nanfang Hospital, Guangzhou First People’s Hospital, Guangzhou Provincial People’s Hospital, SUN Yat-Sen Memorial Hospital, Zhongshan City People’s Hospital and Third Affiliated Hospital of SUN Yat-Sen University)Screening criteriaAllo-HSCT recipients with BOS diagnosed less than 6 monthsStudy durationThe enrolment of the study will start from September 2014 and end at December 2017Control groupPrednisone (initially at 1 mg/kg/day for 4 weeks, and then gradually tapered by 10% of the total dose per week);Azithromycin (250 mg daily in a 4-week cycle for a total of 3 cycles)Experimental groupMSCs (1×106 cells/kg once weekly for 4 consecutive weeks as a cycle; a total of 2 cycles given 2 weeks apart);Prednisone (initially at 1 mg/kg/day for 4 weeks, and then gradually tapered by 10% of the total dose per week);Azithromycin (250 mg daily in a 4-week cycle for a total of 3 cycles)Primary endpointResponse rate at 3 months, defined as the proportion of patients who achieved FEV1 improvement (an increase in FEV1) or steroid sparing (at least 50% dose reduction without FEV1 progression).Secondary endpointsTreatment effect on FEV1 evolution, the incidence of infections and leukemia relapse, safety, overall survival (OS), and leukemia-free survival (LFS).Study proceduresThis is a phase I/II, multicenter, open-label, prospective cohort study. A total of 60 subjects with BOS meeting the inclusion criteria is required. After giving written informed consent, all subjects will choose to receive treatments either MSCs plus azithromycin and prednisone or azithromycin and prednisone alone. Spirometry was performed to evaluate FEV1 at enrollment, 1 and 3 months. Subjects will receive safety assessments after every cycle of treatment.Statistical analysis60-62 subjects will be enrolled to MSC and non-MSC groups at a 1:0.6-1 sample allocation scheme. Response rate will be compared between groups. All AEs of the study will be analyzed.1. BackgroundBronchiolitis obliterans syndrome (BOS) is the most common late noninfectious pulmonary complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), leading to progressive airflow obstruction. Clinical symptoms include cough, dyspnea and wheeze, but patients often remain asymptomatic before moderate to severe decline of airflow being noted in pulmonary function tests (PFT) for months to years. Multiple studies have shown that decline in forced expiratory volume in 1 s (FEV1) is associated with high morbidity and mortality after allo-HSCT. Historically, only 44% and 13% of BOS patients are expected to survive 2 and 5 years, respectively. The pathogenesis of BOS after allo-HSCT is still obscure, but has been suggested in close association with chronic graft-versus host disease (cGVHD), in which ongoing allo-immune attack to host bronchioles. The diagnosis of BOS is mainly based on clinical symptoms and PFT abnormalities. FEV1 is the primary parameter used to grade and follow up BOS patients, as recommended by 2014 National Institutes of Health (NIH) Consensus for cGVHD. ?To date, the standard treatment of BOS after allo-HSCT is still absent. Steroids have been the mainstay of the first-line treatment of BOS, but the efficacy was reported in only 8% to 20% patients. Prolonged systemic steroid exposure confers many severe side effects, especially infection and malignancy relapse, adding to risk of death in BOS. Current guidelines for BOS therapy emphasize the need to evaluate alternative strategies with less toxicity for established BOS. Macrolide antibiotics, azithromycin, have antibiotic and anti-inflammatory properties, and hence are routinely recommended for BOS. Several studies, including ours, suggested that azithromycin combined with steroids could stabilize lung function in some BOS patients. Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitors that can be isolated from various adult tissues, including bone marrow and lung. They possess the capacity to home to sites of injury, modulate immune or inflammation response, and repair tissue. These outstanding properties make MSCs become a promising cell therapy in inflammatory and immune-mediated disorders, such as systemic lupus erythematosus, multiple sclerosis, and chronic lung disease. Of these, the most successfully clinical application of MSCs is in the field of GVHD. We have previously demonstrated that MSCs might be effective for patients with steroid-resistant cGVHD in a single-arm pilot study, and investigated that 5 of 6 patients with lung involvement improved their lung function. Recently, potential benefits of MSCs for BOS were demonstrated by Chambers et al. They reported that the rate of FEV1 decline slowed from 120 to 30 ml/month in the MSC-treated BOS patients after lung transplant, who were unresponsive to prior azithromycin in an open-label single-arm study. In particular, several preclinical studies have provided rationales for use of MSCs in BOS. Raza et al. reported that MSC therapy attenuated peribronchiolar inflammation and fibrosis, and subsequently improved lung histology and PFT of murine BOS after bone marrow transplantation, owing to increasing regulatory T cells and controlling macrophage activation and polarization. Our previous study suggested that MSC-induced amelioration of refractory cGVHD was accompanied by a significantly increased number of interleukin (IL)-10-producing CD5+ B cells. Based on these, we speculate that MSCs may help arrest BOS progression and design this prospective multicentre trial to assess the efficacy and safety of MSCs in BOS following allo-HSCT.2. Objectives2.1 Primary ObjectiveThe primary objective of this study is to evaluate the efficacy of MSCs in patients with BOS after allo-HSCT. 2.2 Secondary ObjectiveThe secondary objective of this study is to evaluate the effect on FEV1 decline, the incidence of infection and leukemia relapse, safety, overall survival, and leukemia-free survival (LFS).3. Study DesignThis is a phase I/II, multicenter, open-label, prospective cohort study of treatment with MSCs plus azithromycin and prednisone (MSC group) compared to treatment with azithromycin and prednisone alone (non-MSC group) in allo-HSCT recipients with BOS diagnosed within 6 months.Screening:The subjects or their legally acceptable representative will provide written informed consent. Besides all examinations detailed in the primary study, high-resolution computed tomography (HRCT), upper respiratory tract viral screen, sputum culture, blood culture, Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-DNA, and peripheral B-cell subsets will be performed to determine baseline status and study eligibility. Bronchoalveolar lavage (BAL) will be performed in case of suspected infection. Lung biopsy is generally not required but could consider on an individual basis.Treatment:Based on patient preferences, subjects will receive MSCs (1×106 cells/kg once weekly for 4 consecutive weeks as a cycle; a total of 2 cycles given 2 weeks apart) plus azithromycin (250 mg daily in a 4-week cycle for a total of 3 cycles) and prednisone (initially at 1 mg/kg/day for 4 weeks, and then gradually tapered by 10% of the total dose per week) (MSC group), or azithromycin and prednisone alone (non-MSC group).4. Subject Selection Criteria4.1 Subject Selection Criteria4.1.1 Number of Subjects A total of 60 subjects will be enrolled to the MSC or non-MSC group.4.1.2 Inclusion Criteria The subjects must meet the following criteria to be eligible for study entry:BOS diagnosis after allo-HSCT for less than 6 months.Diagnosis is based on modified National Institutes of Health (NIH) criteria: (1) FEV1 / vital capacity ＜0.7, and (2) FEV1 ＜75% predicted along with an absolute decline ≥10% from the pre-transplantation baseline, and (3) absence of lower respiratory tract infection.BOS severity is defined as mild (FEV1 60-79%), moderate (FEV1 40-59%), and severe (FEV1 ≤ 39%), according to NIH FEV1-based lung score.Signed Informed Consent FormAged 18 years or above Ability to comply with study and follow-up procedures4.1.3 Exclusion CriteriaThe participant is ineligible if he or she meets ANY one of the following criteria:Uncompensated heart failure, dilated cardiomyopathy Previous treatment with MSCs for GVHDPrior treatment with azithromycin within the past 3 months and total duration of therapy exceeds 1 month Respiratory infection not responding to appropriate treatmentBaseline post-bronchodilator FEV1＜20% predicted or ＞75% predicted Contraindications to steroids (osteonecrosis, osteoporosis, gastric and duodenal ulcers, uncontrollable hyperglycaemia or hypertension, et al) Abnormal pre-transplantation spirometry parametersHistory of pre-HSCT lung disease such as chronic obstructive pulmonary disease, asthma, bronchodilationLeukemia relapse Life expectancy of less than 3 monthsAny conditions not suitable for the trial as judged by the study investigator4.2. Removal CriteriaSubjects who have participated in the study will be removed from statistical analysis for any of the following:1. Subjects who are ineligible for this study2. Deviation(s) from the protocol4.3. Withdrawal CriteriaSubjects will be withdrawn from study treatment for any of the following:Inability to fully comply with the study protocolOccurrence of FEV1 rapidly lost for more than 10% predicted from baseline at 1 month, thought to be due to BOSInitiation of alternative BOS treatment, which will influence the assessment of responseUnacceptable toxicity or uncontrollable infectionLeukemia relapsePatients refused to continue the study at any time after enrollmentBest interest of the subject based upon the treating physician’s discretionThe reason for withdrawal from study participation and the date must be documented in the CRF.5. Study Procedures5.1 Treatment SelectionPatients who meet the eligibility criteria and consent to participate will be given the options of treatment, either MSC plus prednisone and azithromycin or prednisone and azithromycin alone. Patients chose their own treatment procedure after discussion with their treating physicians. 5.2 Study Treatment5.2.1 MSC GroupdrugdosefrequencyroutetimeMSCs1×106 cells/kgonce weeklyIntravenous4 weeks as a cycle; a total of 2 cycles given 2 weeks apartPrednisone1 mg/kgOnce dailyIntravenouskept constant for 4 weeks and then gradually tapered by 10% of the total dose per weekAzithromycin250 mgOnce dailyoralin a 4-week cycle for a total of 3 cycles5.2.2 Non-MSC GroupdrugdosefrequencyroutetimePrednisone1 mg/kgOnce dailyIntravenouskept constant for 4 weeks and then gradually tapered by 10% of the total dose per weekAzithromycin250 mgOnce dailyoralin a 4-week cycle for a total of 3 cycles6. Efficacy Assessments6.1 DefinitionResponse rate, defined as the proportion of patients who achieved FEV1 improvement or steroid sparing.FEV1 improvement, defined as a sustained, measurable increase in FEV1.Steroid sparing, defined as at least 50% dose reduction without FEV1 progression.Disease progression, defined as FEV1 decrease by 10% predicted or more compared with the baseline values.Infection, defined as new-onset clinical signs, such as fever, an elevated C reactive protein, positive cultures (upper respiratory tract viral screen, sputum, blood, BAL, or another site) or radiology, and the clinically identified need to administer antimicrobial drugs.Leukemia relapse, defined as hematologic relapse that were re-appearance of blasts in the peripheral blood, any manifestation of leukemia outside the hematopoietic system, or >5% blasts in the bone marrow smear.Overall survival, defined as the time from BOS diagnosis to death or last contact. Time of BOS diagnosis will use the date of the first PFT that showed BOS.Leukemia-free survival (LFS), defined as the time in continuous complete remission without leukemia relapse.6.2. Primary EndpointThe proportion of patients who achieved response at 3 months6.3 Secondary Endpoints The treatment effect on FEV1 declineIncidence of infections and leukemia relapseSafetyOverall survivalLFS6.4 Schedule and Methods of Endpoints AssessmentsSpirometry including FEV1, HRCT, and microbiological cultures will be performed at enrollment, 1 and 3 months after treatment start. Spirometry will be performed according to American Thoracic Society guidelines. Predicted values for FEV1 will be calculated using published formulae.7. Safety7.1 Safety Endpoints7.1.1 Vital signs and physical examination The vital signs and physical examination must be performed before treatment of every cycle.heart rateblood pressurebody temperaturerate of Respirationsigns of infectionsigns of extrathoracic cGVHD7.1.2 Clinical symptomsThe clinical symptoms include the symptoms may be associated with MSC infusion reported previously. hypertensionpalpitationoral malodorphlebitispruritus7.1.3 Laboratory abnormalities: Peripheral artery blood oxygen saturation during MSC infusion will be monitored by pulse oximetry.Hematology: white cell counts and differential, hemoglobin, and platelet countsHepatic function: Total bilirubin (both direct bilirubin and indirect bilirubin must be documented when the total bilirubin elevates), alanine aminotransferase, aspartate aminotransferase, lactic dehydrogenase, alkaline phosphatase, albumin and total proteinRenal function: urea nitrogen, serum creatinineElectrolytes: sodium, potassium, calcium, and magnesiumUrinalysis: protein, glucose and erythrocyte7.2 Toxicity Assessment of Adverse Events (AE) and Serious Adverse Events (SAE)The investigator is required to make an assessment of the toxicity grade of each AE or SAE reported. In this protocol, the toxicity grade of each AE/SAE will be evaluated according to the Common Toxicity Criteria for Adverse Events (CTCAE) version 3.0.8. Adverse EventsThe investigator or site staff will be responsible for detecting, documenting and reporting events that meet the definition of an AE or SAE as outlined in Section 8.1 and Section 8.3. 8.1 Definition of an AEAny untoward medical occurrence in a subject, temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product. For marketed medicinal products, this also includes failure to produce expected benefits, abuse, or misuse.All AEs and the toxicity grade of each AE/SAE according to CTCAE version 3.0 must be documented in the CRF. Clinical monitors must collect and verify detailed information of AEs when examining original medical records. All AEs should be followed up until resolved. AEs which will not be resolved during clinical observation such as loss of sight, neurotoxicity and deficiency of limbs are graded as unsolved AEs, the blank for resolved time of which don’t need to be filled in the CRF.8.2 Relationship of AEsThe investigator must assess the relationship between AEs and study treatment or concomitant drugs according to the following five categories:Absolutely related: the occurrence of AEs is in accord with chronological sequence after medical procedure and known side effects of suspected drugs, which can resolve after suspension of the drugs and reoccur after reusing of the drugs.Possibly related: the occurrence of AEs is in accord with chronological sequence after medical procedure and known side effects of suspected drugs, which can also occur due to the subject’s disease status or other treatment methods.Possibly unrelated: the occurrence of AEs is not in accord with chronological sequence after medical procedure or known side effects of suspected drugs, which can also occur due to the subject’s disease status or other treatment methods.Unrelated: the occurrence of AEs is not in accord with chronological sequence after medical procedure but in accord with known side effects of non-study treatment drugs. The AEs can also occur due to the subject’s disease status or other treatment methods, disappear after improvement of the disease and suspension of other treatment and reoccur after repeating other treatment methods.Unevaluated: there is no specific relationship between the occurrence of AEs and chronological sequence after medical procedure. The type of AEs is similar to known side effects of suspected drugs, which can also occur due to other drugs.AEs which are in accord with the categories number 1, 2 and 5 above should be documented as AEs of this study.The incidence of AEs=Number of subjects who develop AEs/total subjects×100%8.3 Serious Adverse Events8.3.1 DefinitionA serious adverse event is any untoward medical occurrence that, at any dose:Results in deathIs life-threateningRequires hospitalization or prolongation of existing hospitalizationResults in disability/incapacityHospitalization or prolongation of existing hospitalization for social reasons, examination or other treatment appointed prior to enrollment and events related to BOS progression will not be reported as an SAE. 8.3.1 Reporting of Serious Adverse EventsAll SAEs will be reported promptly in the SAE reporting tables. Initial reports of all SAEs regardless of relationship to study treatment must be submitted to Nanfang Hospital Ethics Committee (phone number: 020-62787926) within 24 hours by the investigator and the follow-up information on previous reports must be submitted as soon as possible, including outcome of SAEs. If the SAE is death, the follow-up information on previous reports is not necessary.9. Rules of Withdrawal9.1 Subjects withdraw from the studySubjects can withdraw from the study at any time for any reason without impact on the investigator’s right to treat disease for subjects. Based upon the interest of subjects, the investigator has the right to request subjects to withdraw from the study for any reason including concomitant disease, adverse events or treatment failure. The core group of clinical study reserves the right to request subjects to withdraw from the study for deviation(s) from the protocol, administrative reasons, or other effective or ethical reasons.The last assessment for subjects must be performed and documented in the CRF regardless of the time and reason for withdrawal. The reason for withdrawal from study participation must be documented in the CRF. All documents related to subjects should be completed. Despite withdrawal from the study, those subjects should be followed up and documented about their diseases until withdrawal of informed consents. For subjects who withdraw from the study due to concomitant diseases or adverse events, the details must be documented in the CRF with other appropriate and valuable data attached.9.2 Premature Termination of the StudyReasons for premature termination of the study include external events, repetition of SAEs, growing incidence of treatment-related death and slow enrolment in the study. All subjects will be informed of premature termination of the study by written consents. Any subjects who decide to discontinue participating in the study must report to the principal investigator.10. Rules of Follow-Up10.1 Follow-up Period Starting from enrollment10.2 Visit SchedulingAt 1 and 3 months after treatment start, or any intervals for reasons such as recurrence or progression of lung manifestations during the follow-up. If the patients in whom an infection is identified, the visit should be postponed for at least 1 month to allow resolution of infection. 10.3 ContentsBesides of the contents detailed in the primary study, the contents of every follow-up visit include complaints of subjects, physical examination, NIH lung symptom scoring, concomitant medication, hematology, peripheral B cell subset analysis, urinalysis, biochemistry and viral loads in blood (EBV-DNA or CMV-DNA) will be also performed. All of the results must be documented in the original medical record.11. Data Analysis and Statistical Considerations11.1 HypothesesThe primary endpoint is the response rate at 3 month. The null and alternative hypotheses are designed with the goal of demonstrating the superiority of the addition of MSCs. Superiority will be determined using the following hypothesis:H0: response rates for MSC and non-MSC group are the same.H1: response rates for MSC and non-MSC group are not the same.11.2 Study Design Considerations This phase I/II, multicenter, open-label, prospective cohort study investigates the efficacy and safety of prednisone and azithromycin combined with or without MSCs in allo-HSCT recipients with BOS diagnosed within 6 months. The primary Endpoint is the response rate at 3 months, and the study is designed to determine if the treatment with MSCs is superior to the treatment without MSCs in the study population. 11.2.1 Sample Size AssumptionsThe sample size calculation is based on the primary endpoint, response rate, with the following assumptions:response rate for the MSC group: 80%. This is based on our previous data from a single-center pilot study, which showed that 5 of 6 cGVHD patients with lung involvement improved their lung function (Peng Y, et al. Leukemia. 2015;29[3]:636-46).response rate for the non-MSC group: 50%This is based on our experience of primary institution in Chinese people (Wu T, et al. Chin J Hematol. 2010;31[2]:114-5).a 1:0.6-1 sample allocation schemea 5% 1-tailed risk of erroneously claiming a difference in the presence of no true underlying difference by χ2 test or Fisher’s exact testa 80% chance of successfully declaring a difference in the presence of a true underlying difference (power)Under the above assumptions, a total sample size of 60-62 subjects is required.The sample size was estimated using a difference test for two proportions on the basis of the primary endpoint. Based on previous data of our single-center pilot study [26], we initially calculated that 30 patients in each study group would be needed to have 80% power to detect a 30% difference in response with a one-side type I error at 0.05. We increased this prespecified sample size on the basis of preliminary experiments, in which response was 67% and 40% in the MSC and non-MSC group, respectively. Then we decided that a sample size of 84 participants would be needed to show significance (patients in preliminary experiments were also included in this study). However, enrollment in the non-MSC group was slower than anticipated, because more patients were willing to receive MSCs therapy. The study was closed with 49 patients in the MSC group and 32 in the non-MSC group. A post-hoc power analysis indicated that this final sample size would be adequate to detect a 28% difference. 11.2.2 Primary Efficacy EndpointThe primary endpoint is the response rate at 3 months.11.2.3 Secondary Efficacy EndpointsThe secondary efficacy endpoints of this study include the treatment effect on FEV1 evolution, the incidence of infection and leukemia relapse, safety, overall survival, and LFS.11.3 Data Analysis ConsiderationsAll collected data will be checked by two physicians for completeness and plausibility, and data analysis will be conducted according to the prespecified statistical analysis plan.Descriptive summaries for continuous data will include the mean, standard deviation, median, minimum, and maximum. Descriptive summaries of discrete data will include the frequency and percentage of each category, as well as overall frequency and percentage totals.11.3.1 Baseline DataBaseline characteristics will be summarized and described in a frequency list.11.3.2 Analysis of efficacy For each numeric variable, the normality of distribution will be preliminarily assessed by the Kolmogorov-Smirnov test. Summary statistics, including proportions, mean, medians, and ranges, are used to describe the patient baseline demographic characteristics, transplantation variables, and adverse events. χ2 test and Mann-Whitney U test are used for categorical variables and continuous variables, respectively. Wilcoxon signed-rank test was used to compare the data obtained before and after treatment.The number and proportion of patients in each group who achieve response will be provided and compared using χ2 test. A logistic regression model will be used to calculate odds ratio and 95% confidence intervals adjusting for factors that reported to influence FEV1 evolution and prognosis, including timing of BOS onset (BOS within 1 year of HSCT or not), BOS severity at enrollment, and the baseline rate of FEV1 decline pre-treatment. The rate of FEV1 decline will be estimated using linear regression squares, which was done by FEV1 values as the dependent variable and time as the independent variable for each subject. The time frame chose to calculate baseline FEV1 rate of decline pre-treatment is within 9 months before enrollment. A minimum of two FEV1 values performed at least 30 days apart were taken to characterize each subject. To evaluate the effect of treatments on FEV1 decline, the difference for change in FEV1 rate of decline between at 3 months and baseline will be compared in univariate and multivariable regression models with adjustment for timing of BOS onset and BOS severity at enrollment. Survival curves will be produced using Kaplan-Meier method and compared using log-rank test. A Cox proportional hazards analysis will be performed to estimate hazard ratios for death.11.3.3 Analysis of safetyAll the patients in the study will be included in the safety analysis. Categorical data such as AEs and SAEs will be summarized by frequency and proportion of total subjects, which will be compared using χ2 test or Fisher’s exact test between MSC and non-MSC group. Quantitative data such as laboratory tests will be described using arithmetic average or median for central tendency and standard deviation or interquartile range for distribution range, which will be compared using t test or non-parametric test between the two groups.12. Materials for the StudyAll materials provided to study sites and investigators are as follows:The study protocolInformed consentCRF13. Ethical Considerations13.1 Responsibility of InvestigatorsThe investigators have the responsibility for guarantee of the clinical study’s compliance with the protocol, Chinese good clinical practice (GCP) guidelines and applicable laws and regulations.13.2 Informed Consent ProcessSubjects must be informed about objectives, methods, possible benefits, potential risks and possible discomforts of the study by investigators before participation in the study. They should be informed that they can withdraw from the study at any time, and there is no impact on the treatment of the disease whether they take part in the study. Subjects or their legally acceptable representative should have enough time to read the inform consent and raise queries. Written informed consent must be obtained from each subject, or their legally acceptable representative.13.3 Protection of Subjects’ Personal DataData collected in the study are limited to the efficacy and safety related to study treatment. Data will be collected and used in accordance with applicable laws and regulations.14. Administrative RequirementsAny revisions of the protocol must be agreed by the investigator and the applicant. To insure the integrity, accuracy and reliability of the data, relevant results of examination and treatment must be documented in original medical record and CRF. Independent clinical monitoring was performed regularly by a panel of qualified and experienced study investigators composed of hematologists who are blinded as to the treatment assignments.15. Quality Control of Data (the same as the primary study) ................
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