Background: A 12-year-old girl diagnosed with intermediate risk B-ALL in 2017 received chemotherapy according to CCLG-2008 ALL protocol and achieved a complete remission (CR) after induction. However, four years later, she had a first bone marrow relapse and received treatment with HKPHOSG Relapsed ALL 2007 protocol. During maintenance chemotherapy, approximately one year after the first relapse, she developed a second bone marrow relapse with NT5C2 gene mutation detected. Tumor burden was not well controlled after DEAV chemotherapy, with the blasts in bone marrow increasing from 49.3% to 96%. The analysis of high-throughput drug sensitivity of tumor resistant genes was consistent with the poor response to chemotherapy. Procedure: CAR-T cell immunotherapy bridged to HSCT was introduced at this stage. Following peripheral lymphocyte apheresis, the patient received lymphodepleting conditioning with fludarabine and cyclophosphamide three days before CAR-T cell infusion. Dexamethasone and carfilzomib were then given according to the outcome of high-throughput drug sensitivity test. Result: Around 68.6% blasts were detected by flow cytometry at the day of CAR-T cell infusion. The patient experienced grade 1 CRS without ICANS. CR of morphology and molecule biology was achieved on day 28 after CAR-T cell infusion. Finally the child received haploid-HSCT and remained in remission. Conclusion: Overall, this report reveals that the combination of drug sensitivity test with lymphodepleting conditioning could significantly reduce the tumor burden before infusion of CART cells, and patients may achieve deeper remission and obtain opportunity for transplantation.

Background: Next-generation sequencing (NGS) is an emerging technology that can comprehensively assess the diversity of the immune system. We explored the feasibility of NGS in detecting minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) based on immunoglobulin and T cell receptor. Methods: Bone marrow samples were collected pre- and post-treatment with pediatric ALL admitted to Shenzhen Children’s Hospital from February 1st, 2020 to January 31st, 2021. We analyzed the MRD detected by NGS, multiparametric flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR), and analyzed risk factors of positive NGS-MRD at the end of B-ALL induction chemotherapy. Results: A total of paired 236 bone marrow samples were collected from 64 children with ALL (58 B-ALL and 6 T-ALL). The decrease in the clonal rearrangement frequency of IGH, IGK, and IGL was generally consistent after treatment. Positive MRD was detected in 57.5% (77/134) of B-ALL and 80% (12/15) of T-ALL by NGS after chemotherapy, which was higher than those detected by MFC and RQ-PCR. In B-ALL patients, MRD results detected by NGS were consistent with MFC（r = 0.708, p < 0.001）and RQ-PCR（r = 0.618, p < 0.001）. At the end of induction, NGS-MRD of 40.4% B-ALL was >0.01% and multivariate analysis indicated that ≧2 clonal rearrangement sequences before treatment were an independent factor of negative NGS-MRD. Conclusions: NGS is more sensitive than MFC and RQ-PCR for MRD measurement. B-ALL children with ≧2 clonal rearrangements detected by NGS before treatment are difficult to switch to negative MRD after chemotherapy.