2 Methods
Between September 2017 and July 2020, 8 cases of CD19+ B-ALL pediatric patients relapsed with both bone marrow and extramedullary infiltrations and enrolled in this study. The study was conducted in Shanghai Children’s Medical Center affiliated to Shanghai Jiao Tong University School of Medicine and Children’s Hospital of Soochow University and was registered at chictr.org.cn (ChiCTR2100041852). Informed consent form was signed by the guardians of patients, which required the admission of the ethics committee of Shanghai Children’s Medical Center and Children’s Hospital of Soochow University.
Peripheral blood (PB) was directly drawn from patients or their donor to gain T cells positively selected by anti-CD3-coated beads (Invitrogen). The manufacture of CAR-T cells usually took 7~10 days. Prior to CAR-T injection, all patients were administered with lymphodepleting chemotherapy: 500mg/m2cyclophosphamide for 2 days (day-4, -3) and 40mg/m2fludarabine for 3 days (day-4, -3, and -2). At day0, patients were infused fresh CAR-T cells with the dosage of 1~15×106/Kg. Patients with severe infection were postponed the CAR-T infusion till there was no fever for 24 hours, as they were infused with cryopreserved CAR-T cells. Patients who relapsed after the first exposure to CAR-T cells were allowed to receive a second CAR-T, and administered with 500mg/m2cyclophosphamide and 50mg/m2 fludarabine per day for 3 days (day-4, -3, and -2) for lymphodepletion.
Neurologic adverse events were graded according to CTCAE 4.03, and cytokine release syndrome (CRS) was graded as per the American Society of Transplant and Cellular Therapy grading criteria15. A bone marrow aspiration was done after 7~14 days following CAR-T infusion, and disease burden assessment was performed by morphology, flow cytometry (FCM) based MRD monitoring. A negative status for MRD from bone marrow samples was defined as less than 0.01% of abnormal cells identified by multiparameter flow cytometry performed at Key Laboratory of Hematology and Oncology Ministry of Health. Imaging of the relevant extramedullary sites was performed at 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3years following CAR-T cell infusion. CAR-T cell persistence in peripheral blood or CSF of patients was measured by the real time qPCR and copies per ug DNA was normalized by the single-copy gene, CDKN1a.
For event-free survival (EFS), an event was defined as bridging to HSCT, relapse or death. Death was the event for analysis of overall survival (OS). Kaplan-Meier curves were created for EFS and OS analysis and compared with the log-rank test. All statistics were performed as indicated using Graph-Pad Prism 8 software for Windows, version 8.0.1. Statistically significant differences were defined at a P value <0.05.