Ren-Yi Su, Sun-Bin Ling, Qiao-Nan Shan , Xu-Yong Wei , Rui Wang , Chang-Ku Jia , Li Zhuang , Tian Shen , Li-Min Ding , Zhi-Dan Xu , Lai-Bang Luo , Li-Bo Sun, Guang-Ming Li , Tai-Shi Fang , Nan Jiang , Kun Zhang , Zhao-Jie Su , Zhi-Hai Peng , Ren Lang , Tao Jiang , Qiang He , Lin-Sen Ye 0 , Yang Yang 0 , Yu-Ting He , Wen-Zhi Guo , Liu-Gen Lan , Xu-Yong Sun , Dong Chen , Zhi-Shui Chen , Da-Wei Zhou , Shao-Jun Ye , Qi-Fa Ye , Min Tian , Jian-Hua Shi , Bo Wang , Jiang Liu , Qian Lu , Wei Rao , Jin-Zhen Cai , Tao Lv , Jia-Yin Yang , Pu-Sen Wang , Lin Zhong , Jing-Sheng Ma 0 , Qi-Gen Li 0 , Sheng-Dong Wu , Chang-Jiang Lu , Cai-De Lu , Dong-Hua Zhang , Xuan Wang , Zi-Qiang Li , Mu-Jian Teng , Jun-Jie Li , Wen-Tao Jiang , Jian-Hua Li , Quan-Bao Zhang , Ning-Qi Zhu , Zheng-Xin Wang , Kang He , Qiang Xia , Shao-Hua Song , Zhi-Ren Fu , Wei Qiu , Guo-Yue Lv , Rui-Peng Song , Ji-Zhou Wang , Zheng Wang 0 , Jian Zhou 0 , Gang Chen , Ying-Peng Zhao , Li Li , Ze-Min Hu , Qi-Jie Luo , Zhong-Zhou Si , Bin Xie , Xiao-Shun He , Zhi-Yong Guo , Shu-Sen Zheng , , , , Xiao Xu , , ,

1 Department of Hepatobiliary and Pancreatic Surgery, The Center for Integrated Oncology and Precision Medicine, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310 0 06, China

2 NHC Key Laboratory of Combined Multi-organ Transplantation, Hangzhou 310 0 03, China

3 Department of Hepatobiliary and Pancreatic Surgery, Shulan (Hangzhou) Hospital, Zhejiang Shuren University School of Medicine, Hangzhou 310022, China

4 Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310 0 03, China

5 Department of Transplantation, Jiangxi Provincial People’s Hospital Affiliated to Nanchang University, Nanchang 330 0 06, China

6 Liver Transplantation Center, Beijing You’an Hospital, Capital Medical University, Beijing 10 0 069, China

7 Department of Hepatic Surgery, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital; The Second Affiliated Hospital of Southern University of Science and Technology, Shenzhen 518112, China

8 Department of General Surgery, Xiang’an Hospital of Xiamen University, Xiamen 3610 0 0, China

9 Deartment of Hepatobiliary and Pancreaticosplenic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 10 0 020, China

10 Department of Hepatic Surgery and Liver Transplantation Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China

11 Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

12 Department of Liver Transplantation, The Second Affiliated Hospital of Guangxi Medical University, Nanning 530 0 0 0, China

13 Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

14 Institute of Hepatobiliary Diseases of Wuhan University, Transplant Center of Wuhan University, Hubei Key Laboratory of Medical Technology on Transplantation, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

15 Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China

16 Liver Transplantation Center, Tsinghua Changgung Hospital, Beijing 102218, China

17 Organ Transplantation Center, Affiliated Hospital of Qingdao University, 59 Haier Road, Laoshan District, Qingdao 266061, China

18 Department of Liver Surgery, Liver Transplantation Center, West China Hospital, Sichuan University, Chengdu 610044, China

19 Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20 0 080, China

20 Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang 330 0 06, China

21 Department of Hepatobiliary and Pancreatic Surgery, Ningbo Medical Center Lihuili Hospital, Ningbo University, Ningbo 315041, China

22 Liver Transplant Center, General Hospital of Eastern Theater Command, Nanjing 210 0 02, China

23 Department of General Surgery, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Jinan 250014, China

24 Liver Transplant Department, Tianjin First Center Hospital, Tianjin 300192, China

25 Department of General Surgery, Huashan Hospital, Fudan University, Shanghai 20 0 040, China

26 Department of Liver Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

27 Liver Transplantaiton Center, Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 20 0 025, China

28 Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Changchun 130021, China

29 Department of Hepatobiliary Surgery, Anhui Province Key Laboratory of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of USTC; Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230 0 01, China

30 Department of Liver Surgery & Transplantation, Zhongshan Hospital Fudan University, Shanghai 20 0 032, China

31 Department of hepato-biliary-pancreatic surgery and liver transplantation center, the First People’ s Hospital of Kunming, Kunming 650 0 0 0, China

32 Department of Hepatobiliary Surgery, Zhongshan City People’s Hospital, Zhongshan 528499, China

33 Department of Liver Transplantation Center, The Second Xiangya Hospital of Central South University, Changsha 410011, China

34 Department of Hepatic Surgery and Liver Transplantation Center, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510062, China

Keywords: Sirolimus Immunosuppressive agents Hepatocellular carcinoma Liver transplantation Conversion

ABSTRACT Mammalian target of rapamycin (mTOR) inhibitor as an attractive drug target with promising antitumor effects has been widely investigated. High quality clinical trial has been conducted in liver transplant (LT) recipients in Western countries. However, the pertinent studies in Eastern world are paucity. Therefore, we designed a clinical trial to test whether sirolimus can improve recurrence-free survival (RFS) in hepa- tocellular carcinoma (HCC) patients beyond the Milan criteria after LT. This is an open-labeled, single-arm, prospective, multicenter, and real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in HCC patients after LT. Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center-specific for the first 4-6 weeks. The following regimens integrated sirolimus into the regimens as a combination therapy with reduced calcineurin inhibitors based on the condition of patients and centers. The study is planned for 4 years in total with a 2-year enrollment period and a 2-year follow-up. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indicator RFS as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator of overall survival. We hope that the outcome will provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese popula- tion data.

Introduction

Hepatocellular carcinoma (HCC) is a common malignancy with ever-growing morbidity and substantial mortality worldwide [1] . It is estimated that over half of all HCC cases and HCC-related deaths occur in China [2] . Liver transplantation (LT) is one of the most ef- fective life-saving treatments for HCC patients [3] . Compared with other surgical treatment options such as resection and ablation, LT takes its grips on conferring higher long-term survival benefits [4] .

The immunosuppressive drugs ushered in a new dawn in the field of LT with properties of anti-rejection. However, a caveat of immunosuppression resides in the double-edged sword effects that not only reduces immunological rejection, but also adds to the risk of HCC recurrence and other complications compromising the long-term outcomes in liver recipients. Calcineurin inhibitors (CNIs), namely tacrolimus and cyclosporine, are widely used in clinical practice. Protumorigenic property is one of the primary issues of CNIs therapy. In LT rat model, both cyclosporine and tacrolimus have been shown to enhance liver tumor invasiveness and recurrence [ 5 , 6 ]. Clinically, the result is consistent with pre- clinical study and demonstrates that CNIs increase the risk of HCC recurrence after LT in a dose-dependent fashion [7] . Complica- tion such as renal insufficiency is also increased after LT, which is mainly due to the nephrotoxic effects of CNIs.

An ideal acceptable immunosuppressant should be the one with minimal nephrotoxicity and no pro-tumorigenic effects. Recently, great attention has been bestowed to a new class of immunosup- pressive agent, mammalian target of rapamycin (mTOR) inhibitors. Sirolimus, the only approved mTOR inhibitor in China, is charac- terized with antitumorigenic effects and minimal nephrotoxicity. Mechanically,invivostudy shows that sirolimus significantly re- duces tumor growth and that this effect is mainly mediated by an- tiangiogenic effects [8] . In addition, sirolimus inhibits proliferation and migration phenotypes in HCC mice model via modulation of GP73 [9] . Another rationale is that mTOR pathway is vital in can- cer progressionperse, which has synergistic effects with antian- giogenic property to anti-hepatocarcinogenesis [10] .

From clinical perspective, the phase 3 randomized controlled trial (RCT) named “SiLVER Study” with an 8-year follow-up period performed by Geissler et al. [11] (NCT00355862) demonstrated that sirolimus could provide a benefit of recurrence-free survival (RFS) at 3 years after transplantation, but not at the study end. Subgroup analysis showed that recipients within the Milan criteria signifi- cantly benefit in RFS during early post-LT period compared with recipients beyond the Milan criteria (no benefit across the whole period) [11] . In contrast, our previous study revealed that HCC pa- tients beyond the Milan criteria may benefit from sirolimus [12] . A recent meta-analysis included a total of 11 studies including 1 RCT testified the safety and effectiveness of sirolimus-based immuno- suppression in HCC patients following LT. The pooled results found that sirolimus-administrated recipients had lower recurrence com- pared with the control group [13] .

As the differences exist in etiology, race, indications and proce- dures for LT between Western and Eastern models, evidence from Western studies may not be applicable in Eastern settings. Based on the experimental observations and clinical considerations, we designed a clinical trial in Chinese populations with the purpose to test whether sirolimus can improve RFS in LT patients. We pre- dict an improved RFS in LT patients treated with sirolimus.

Methods/design

Basic protocol overview

This is an open-labeled, single-arm, prospective multicenter, real-world study aiming to evaluate the clinical outcomes of early switch to sirolimus-based regimens in patients undergoing LT for HCC. We have named the trial the “LEAP-Sirolimus (LEAPS)”(ChiCTR210 0 042869) with a double meaning. Firstly, it refers to a combination of the sirolimus and name of our organization, namely Liver Elite Aspiration Network 9Plus. Secondly, “LEAPS”contains our expectation and prediction for giant leaps to be made in this trial. The study is planned for 4 years in total with a 2- year enrollment period and a 2-year follow-up ( Fig. 1 ). Patients with a histologically proven HCC and beyond the Milan criteria will be enrolled. The initial immunosuppressant regimens are center- specific for the first 4-6 weeks. The following regimens integrate sirolimus into the regimens as a combination therapy with reduced CNI based on the condition of patients and centers.

Fig. 1. Study scheme of LEAPS trial. LT: liver transplantation; HCC: hepatocellular carcinoma; HIV: human immunodeficiency virus; mTOR: mammalian target of rapamycin; MPA: mycophenolate mofetil; CNIs: calcineurin inhibitors.

Notably, patients are not prohibited to use antitumor drugs in- cluding traditional chemotherapies, immunotherapies and target drugs, but detailed records of type, dosage and duration of med- ication are warranted.

Inclusion criteria

The trial enrolls all patients eligible for LT as listed by each of the participating centers. The specific inclusion criteria are as follows: age between 18 and 70 years, histologically proven HCC before early switch to sirolimus-based regimens, written informed consent given by the patient. Another inclusion criterion is accep- tance of both deceased liver donors and living liver donors. We only include patients beyond the Milan criteria at the time of LT but without macrovascular invasion (expect for branch of portal vein) and distant metastasis. One caveat is that the latter shall pre- vail when divergences emerge between pre-LT diagnosis and post- LT pathology.

Exclusion criteria

Patients meeting one of the following criteria are excluded: multiple-organ recipients, patients known to be hypersensitive to sirolimus or its derivates, hyperlipidemia refractory to optimal medical treatment (triglyceride ≥400 mg/dL or ≥4.6 mmol/L, to- tal cholesterol ≥300 mg/dL or ≥7.8 mmol/L), evidence of local or systemic infection, human immunodeficiency virus-positive pa- tients, platelets counts＜35 × 109/L, white blood cell counts＜1.5 × 109/L, urine routine: protein ++ or beyond, women who are pregnant, lactating or child-bearing potential not willing to take contraception, patients with history of non-HCC tumor within 5 years prior to inclusion (basal cell and squamous cell skin cancers that have been successfully treated are not included), patients with a psychological, familial, sociologic or geographic condition difficult to comply with study protocol or follow-up schedule, and under guardianship (e.g. individuals who are not able to freely give their informed consent).

Withdrawal criteria

Participants who meet the withdrawal criteria will be with- drawn from the study but not replaced. The withdrawal criteria for this study are as follows: participants who actively request to withdraw from the study (retract/revoke informed consent), lost to follow-up, taking medications prohibited for the trial, with a history of allergic reaction to sirolimus or its additives, pregnant, violating trial protocol, subjects censored at death, clinical cases when physicians in charge believe that continued participation in the study is not in the best interest of the patient considering any pathological event, clinical adverse event, or change in the sub- ject’s physical condition, and other reasons to be withdrawn from the study.

Study objectives

We predict that this sirolimus-based regimen will improve HCC-free survival in LT population. The primary endpoint of this study is the occurrence of metastasis and/or recurrence after LT procedure. Secondary endpoints include tumor-related death, inci- dence of allograft rejection, graft loss and alterations in renal func- tion.

Treatment scheme

Patients are treated with center-specific immunosuppressive protocol for the first 4-6 weeks, excluding use of any mTOR- inhibitor medications. The initial immunosuppressant regimens in- clude CNIs ± mycophenolic acid (MPA) ± glucocorticoid immuno- suppressant ± IL-2 inhibitor (basiliximab) regimen. Tacrolimus is preferred in CNIs drugs. MPA drugs can be Cellcept (MMF) or My- fortic (EC-MPS). Four to ten ng/mL for sirolimus and 50% reduction in CNIs are recommended but not strictly restricted. The choice of regimen and dose of drugs should be based on the condition of patients and centers. Our only requirement is comprising sirolimus conversion regimen including the use of sirolimus starting between day 29 and day 42 after LT and a simultaneous reduction in CNIs.

Follow-up and documentation

All patients enrolled in the study will be followed up at least 7 times in 2 years. Follow-up examinations will be done on second and third month, then every 3 months to the end of first year and every 6 months to the end of second year.

The management of the data in the study is performed using the Electronic Data Capture (EDC) system. Trial data will be col- lected through the electronic case record form (eCRF). Regular doc- umentations include evaluation of symptoms and signs, monitor- ing the trough concentrations of CNIs and sirolimus, patient recur- rence/metastasis information and patient survival, changes in re- nal function, concomitant medication (e.g., type, dosage and du- ration) and adverse event information. Lost-to-follow-up is de- fined as missing two consecutive follow-up visits. If the last con- tact is prior to the diagnosis of a recurrence/metastasis event, recurrence/metastasis-free survival time is censored using the date of the last contact. If patient can be re-contacted, the status should be reset to alive. Notably, recurrence/metastasis-free survival time and corresponding censoring status will remain. If definite date of a patient’s death is documented, the status will be "dead".

Endpoint definition

The definition of HCC RFS is the time interval between the first date of LT and the date of HCC recurrence/metastasis or death; censored for RFS will be made for patients alive or recurrence-free at the time of their last contact. HCC recur- rence/metastasis is defined as either histologically confirmed tu- mor recurrence/metastasis, or unequivocal evidence according to the Barcelona Criteria (Barcelona-20 0 0 EASL Conference). Date of recurrence is defined as the first day of tumor suspicion. Clinical evidence for HCC recurrence includes symptoms and signs (e.g., pruritis and ascites), laboratory examination [e.g., alpha-fetoprotein (AFP) and PIVKA-II], liver image by computerized tomography or magnetic resonance imaging, as well as diagnostic action of Barcelona Criteria. Recurrence of HCC can therefore be evaluated through the whole follow-up period besides the protocol required visits.

Trial organization

The study is an investigator-initiated trial. Preliminary inves- tigator meetings to plan the details of the trial protocol were organized by Shulan (Hangzhou) Hospital and Hangzhou First People’s Hospital. Services for site monitoring, data management and arranging regulatory affairs are provided by experts familiar with the trial in Shulan (Hangzhou) Hospital and Hangzhou First People’s Hospital. Statistical research is performed within Shulan (Hangzhou) Hospital, Hangzhou First People’s Hospital and China Liver Transplant Registry.

Participating centers

Major liver transplant centers from different provinces are par- ticipating this clinical trial. In total, 33 sites covering most parts of China up to 19 provinces have committed to participate the study.

Drug supply

One mg sirolimus (Rapamune) blisters produced by Pfizer Ire- land Pharmaceuticals (Little Connell, Newbridge, Co. Kildare, Ire- land) is used as study medication in participating centers.

Quality control and on-site monitoring

Quality control is performed across whole process of the study including screening and determination of eligibility of research centers, laboratory and researchers to fully meet requirements of the national law “Quality Management Practice for Medical Device Clinical Trials” in China. Recruitment and follow-up of patients, regular monitoring of safety and endpoint data are performed ac- cording to good clinical practice (GCP) guidelines. Data manage- ment is performed by each center and documentations from clini- cal trials are maintained and managed in accordance with the GCP requirements.

Ethics and safety

Protocol version 1 has been reviewed and approved by the re- sponsible Institutional Review Board/Institutional Ethics Commit- tee (IRB/IEC) for all of centers. The study protocol complies with theDeclarationofHelsinkiand principles of the GCP guidelines. Written informed consent is obtained from all participants at en- rollment. Each patient is fully informed about the procedure of the trial and liable reasonable risk by an experienced physician famil- iar with the study. The safety and all potential side effects for the patients are monitored at least once a year by IRB/IEC. Qualified personnel from all sites should also attend meeting regularly to review safety data, including adverse events and serious adverse events. Any information deemed to potentially affect the safety of the trial should be reported to IRB/IEC in time.

Statistical analysis

A one-sided, one-sample log-rank test calculated from a sample of 397 subjects achieves 90.1% power at a 0.025 significance level to detect a median survival time of 1.20-fold in the new group when the median survival time of the historic control group is 1.00. Subjects are accrued for 2.0 years. Follow-up that continues for 2 years after the last subject is added. The probability of a sub- ject experiencing an event during the study is 0.8132. The expected number of events during the study is 323. It is assumed that the survival time distributions of both groups are approximated rea- sonably well by the Weibull distribution with a shape parameter of 1.00.

Demographic and clinical characteristics of participants are summarized using standard descriptive statistics. Time-to-event end points including RFS, overall survival (OS) and tumor-related death are assessed at the end of the study estimated by the Kaplan-Meier method. As part of the secondary analysis, we mainly focus on the alterations in renal function as its potential renal protective effect. Further stratified analyses are performed on the stratification of adjuvant therapy. Safety parameters for inci- dence of adverse events are analyzed based on the safety analysis set which is defined as subjects who receive at least one dose of study drug. All continuous variables are compared by Student’st- test or rank-sum test and rate is compared by Chi-square test.

Discussion

There are two substantial issues in patients undergoing LT pro- cedure for HCC: graft rejection and tumor recurrence. Recent stud- ies have shown that sirolimus bears a potential antitumorigenic ef- fects [ 6 , 9 , 10 ]. The SiLVER study is the first RCT trial which eval- uates HCC recurrence in LT recipients [11] . Results by the confir- matory analysis of the SiLVER-trial in 2016 have drawn three im- portant conclusions. Firstly, sirolimus conversion does not improve long-term RFS beyond 5 years in LT recipients with HCC. Secondly, younger (age ≤60 years), low-risk (within the Milan criteria) re- cipients and patients with sirolimus monotherapy also benefited from sirolimus treatment. Thirdly, no contraindicative overall dis- advantage regarding serious adverse events of sirolimus therapy became apparent during the trial. The trial lays the foundation for applying sirolimus-based immunosuppressive regimen to LT pa- tients with HCC and provides a reasonable clinical rationale for our study. Although sirolimus treatment provides a survival advantage up to 5 years after transplantation, it seems ambiguous for the an- titumor effect as it’s diminishing RFS benefits. More recently, the SiLVER-trial published its data with exploratory approach revealing a clearer antitumor effect [14] . The analysis included data from 508 patients with a follow-up period of 72.4 months on average. Mul- tivariate analysis showed an independent benefit of sirolimus use for ≥3 months for OS. Subgroup analysis demonstrated that the patients deriving the maximum benefit reside in those with AFP ≥10 ng/mL and having used sirolimus for ≥3 months with regard to OS, disease-free survival, and HCC recurrence. Higher tumor ac- tivity in LT patients with AFP evidence is advocated for mTOR in- hibitor use.

Here, we put forward three key issues to consider when design- ing this trial. The first consideration is that our study population only includes LT patients with HCC (beyond the Milan criteria) and without macrovascular invasion (expect for branch of portal vein) and distant metastasis. This trial is a continuation of our previous work that revealed recipients exceeding, rather than meeting, the Milan criteria may benefit from sirolimus [12] . Among recipients beyond the Milan criteria, our unpublished data suggest a signif- icant survival benefit in branch invasion of portal vein compared with major portal vein invasion. And we have observed compara- ble prognosis between the branch invasion of portal vein group and without macrovascular invasion group. Our reasoning is that particular patients beyond the Milan criteria and with branch in- vasion of portal vein may benefit most from sirolimus conversion regimen, similar to the finding that higher tumor activity in LT may benefit from sirolimus use as declared in SiLVER study. Ad- ditionally, choosing RFS as our primary endpoint is another critical point. According to the published data from SiLVER study, encour- aging results demonstrate a clear antitumor effect particularly in patients with AFP ≥10 ng/mL and having used sirolimus for ≥3 months. In our study, we assert a similar benefit of sirolimus in prognosis (e.g., lower recurrence rate, later tumor redevelopment, longer survival time after recurrence). Thus, single-arm trial using sirolimus conversion is preferred after intense debate for ethical considerations. Final issue to consider is that our study is aimed to provide better evidence for clinical decision-making and revising treatment guidelines based on Chinese population data. A limita- tion of the trial is the intrinsic disadvantage of single-arm study. Without simultaneous control, it may undermine the credibility of research as the survival rate of recipients undergoing LT procedure may be higher than the counterpart in previous studies regard- ing the development of antitumor drugs, surgery technology and medical conditions. Essentially, we prospectively assess the clinical value of sirolimus in a real-world setting without prohibiting ad- ditional therapy (e.g., immunotherapies and target drugs) to bet- ter reflect the actual clinical benefits for LT recipients beyond the Milan criteria. We expect to provide multiple immunosuppressant regimens for clinicians and patients in the clinical scenario.

The LEAPS study is the first open-labeled, single-arm, prospec- tive multicenter, real-world trial based on Chinese population that primarily evaluates HCC recurrence in LT recipients beyond the Milan criteria. We predict that sirolimus conversion regimen will provide survival benefits for patients particular in the key indica- tor “RFS” as well as better quality of life. If the trial is conducted successfully, we will have a continued monitoring over a longer follow-up time to estimate indicator “OS”.

Acknowledgments

The authors would like to dedicate this article to the memory of the late Yun-Jin Zang, the esteemed pioneer in the field of liver transplantation for his inspiration and assistance in initiating this clinical trial.

CRediT authorship contribution statement

Ren-YiSu:Conceptualization, Visualization, Writing - original draft.Sun-BinLing:Conceptualization, Writing - review & editing.Qiao-NanShan:Conceptualization, Project administration, Data curation, Writing - review & editing.Xu-YongWei:Conceptualiza- tion, Project administration, Data curation, Writing - review & edit- ing.RuiWang: Methodology, Formal analysis, Software, Writing - review & editing.Chang-KuJia:Conceptualization, Project ad- ministration, Data curation, Writing - review & editing.LiZhuang:Conceptualization, Project administration, Data curation, Writing - review & editing.TianShen:Conceptualization, Project adminis- tration, Data curation, Writing - review & editing.Li-MinDing:Conceptualization, Project administration, Data curation, Writing - review & editing.Zhi-DanXu:Conceptualization, Project adminis- tration, Data curation, Writing - review & editing.Lai-BangLuo:Conceptualization, Project administration, Data curation, Writing - review & editing.Li-BoSun:Conceptualization, Project administra- tion, Data curation, Writing - review & editing, Writing - review & editing.Guang-MingLi:Conceptualization, Project administration, Data curation, Writing - review & editing.Tai-ShiFang:Conceptu- alization, Project administration, Data curation, Writing - review & editing.NanJiang:Conceptualization, Project administration, Data curation, Writing - review & editing.KunZhang:Conceptualiza- tion, Project administration, Data curation, Writing - review & edit- ing.Zhao-JieSu:Conceptualization, Project administration, Data curation, Writing - review & editing.Zhi-HaiPeng:Conceptual- ization, Project administration, Data curation, Writing - review & editing.RenLang:Conceptualization, Project administration, Data curation, Writing - review & editing.TaoJiang:Conceptualiza- tion, Project administration, Data curation, Writing - review & edit- ing.QiangHe:Conceptualization, Project administration, Data cu- ration, Writing - review & editing.Lin-SenYe:Conceptualization, Project administration, Data curation, Writing - review & editing.YangYang:Conceptualization, Project administration, Data cura- tion, Writing - review & editing.Yu-TingHe:Conceptualization, Project administration, Data curation, Writing - review & editing.Wen-ZhiGuo:Conceptualization, Project administration, Data cu- ration, Writing - review & editing.Liu-GenLan:Conceptualization, Project administration, Data curation, Writing - review & editing.Xu-YongSun:Conceptualization, Project administration, Data cu- ration, Writing - review & editing.DongChen:Conceptualization, Project administration, Data curation, Writing - review & editing.Zhi-ShuiChen:Conceptualization, Project administration, Data cu- ration, Writing - review & editing.Da-WeiZhou:Conceptualiza- tion, Project administration, Data curation, Writing - review & edit- ing.Shao-JunYe:Conceptualization, Project administration, Data curation, Writing - review & editing.Qi-FaYe:Conceptualization, Project administration, Data curation, Writing - review & edit- ing.MinTian:Conceptualization, Project administration, Data cu- ration, Writing - review & editing.Jian-HuaShi:Conceptualization, Project administration, Data curation, Writing - review & editing.BoWang:Conceptualization, Project administration, Data curation, Writing - review & editing.JiangLiu:Conceptualization, Project administration, Data curation, Writing - review & editing.QianLu:Conceptualization, Project administration, Data curation, Writing - review & editing.WeiRao:Conceptualization, Project administra- tion, Data curation, Writing - review & editing.Jin-ZhenCai:Con- ceptualization, Project administration, Data curation, Writing - re- view & editing.TaoLv:Conceptualization, Project administration, Data curation, Writing - review & editing.Jia-YinYang:Concep- tualization, Project administration, Data curation, Writing - review & editing.Pu-SenWang:Conceptualization, Project administration, Data curation, Writing - review & editing.LinZhong:Conceptu- alization, Project administration, Data curation, Writing - review & editing.Jing-ShengMa:Conceptualization, Project administra- tion, Data curation, Writing - review & editing.Qi-GenLi:Con- ceptualization, Project administration, Data curation, Writing - re- view & editing.Sheng-DongWu:Conceptualization, Project ad- ministration, Data curation, Writing - review & editing.Chang-JiangLu:Conceptualization, Project administration, Data curation, Writing - review & editing.Cai-DeLu:Conceptualization, Project administration, Data curation, Writing - review & editing.Dong-HuaZhang:Conceptualization, Project administration, Data cura- tion, Writing - review & editing.XuanWang:Conceptualization, Project administration, Data curation, Writing - review & editing.Zi-QiangLi:Conceptualization, Project administration, Data cura- tion, Writing - review & editing.Mu-JianTeng:Conceptualiza- tion, Project administration, Data curation, Writing - review & edit- ing.Jun-JieLi:Conceptualization, Project administration, Data cu- ration, Writing - review & editing.Wen-TaoJiang:Conceptualiza- tion, Project administration, Data curation, Writing - review & edit- ing.Jian-HuaLi:Conceptualization, Project administration, Data curation, Writing - review & editing.Quan-BaoZhang:Concep- tualization, Project administration, Data curation, Writing - review & editing.Ning-QiZhu:Conceptualization, Project administration, Data curation, Writing - review & editing.Zheng-XinWang:Con- ceptualization, Project administration, Data curation, Writing - re- view & editing.KangHe:Conceptualization, Project administration, Data curation, Writing - review & editing.QiangXia:Conceptu- alization, Project administration, Data curation, Writing - review & editing.Shao-HuaSong:Conceptualization, Project administra- tion, Data curation, Writing - review & editing.Zhi-RenFu:Con- ceptualization, Project administration, Data curation, Writing - re- view & editing.WeiQiu:Conceptualization, Project administration, Data curation, Writing - review & editing.Guo-YueLv:Conceptu- alization, Project administration, Data curation, Writing - review & editing.Rui-PengSong:Conceptualization, Project administration, Data curation, Writing - review & editing.Ji-ZhouWang:Con- ceptualization, Project administration, Data curation, Writing - re- view & editing.ZhengWang:Conceptualization, Project adminis- tration, Data curation, Writing - review & editing.JianZhou:Con- ceptualization, Project administration, Data curation, Writing - re- view & editing.GangChen:Conceptualization, Project administra- tion, Data curation, Writing - review & editing.Ying-PengZhao:Conceptualization, Project administration, Data curation, Writing - review & editing.LiLi:Conceptualization, Project administration, Data curation, Writing - review & editing.Ze-MinHu:Conceptu- alization, Project administration, Data curation, Writing - review & editing.Qi-JieLuo:Conceptualization, Project administration, Data curation, Writing - review & editing.Zhong-ZhouSi:Conceptu- alization, Project administration, Data curation, Writing - review & editing.BinXie:Conceptualization, Project administration, Data curation, Writing - review & editing.Xiao-ShunHe:Conceptual- ization, Project administration, Data curation, Writing - review & editing.Zhi-YongGuo:Conceptualization, Project administration, Data curation, Writing - review & editing.Shu-SenZheng:Con- ceptualization, Supervision, Writing - review & editing.XiaoXu:Conceptualization, Supervision, Funding acquisition, Writing - re- view & editing.

Funding

This study was supported by grants from the National S&T Ma- jor Project (2017ZX10203205), Key Program, National Natural Sci- ence Foundation of China ( 81930016 ) and Zhejiang Provincial Nat- ural Science Foundation of China (LY21H160026).

Ethical approval

This study was reviewed and approved by the Institutional Re- view Board/Institutional Ethics Committee in each center.

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the sub- ject of this article.