PMC8530183_01
Process of treatment with CAR-T cells and clinical response. (A) The process of CAR-T-cell treatment and clinical response in the BM. The results of PET-CT are shown in (B-I). (B) At diagnosis, multiple high metabolic lesions were detected in bilateral neck, axilla, mediastinum, abdominal, and retroperitoneal cavity. (C) Before radiotherapy, multiple lesions in the mediastinum and abdominal cavity were involved by EM leukemia, as well as the gastric wall. (D) One month after the 1st CAR-T-cell infusion, extramedullary leukemia achieved full resolution. (E) Four months after the 1st CAR-T infusion, extramedullary leukemia remained at full resolution. (F) Eight months after the 1st CAR-T infusion, the EM leukemia recurred in multiple lesions, including lymph nodes in the mediastinum and right inner mammary region, intrapericardial, bilateral kidney, retroperitoneal, perirenal, and pelvic peritoneum. (G) Twenty-six days after the 2nd CAR-T cell infusion, EM leukemia regressed. (H) Seventy-four days after the 2nd CAR-T cell infusion and 10 days before the 3rd CAR-T infusion, EM leukemia progressed. (I) Three months after the 3rd CAR-T cell infusion, EM leukemia achieved full resolution again. BM, bone marrow; EM, extramedullary; CAR, chimeric antigen receptor. (J) Immunohistochemical results of renal tissue showed weak positive CD22 in tumor cells.
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The treatment progress is shown in  Figure 1A . Unfortunately, the patient underwent EM relapse in the mediastinum 2 years after allo-HSCT ( Figure 1B ). PET-CT revealed multiple lesions in the mediastinum and abdominal cavity involved EM leukemia, with further evidence of gastric wall involvement ( Figure 1C ). EM leukemia full resolution was confirmed 1 month later by PET/CT ( Figure 1D ). However, the MRD positivity in the BM increased to 0.02% at 4 months with EM leukemia free ( Figure 1E ) followed by blasts in the BM and MRD increased to 5.5% (CD19+CD22+) and 6.08% at 8 months after CAR-T-cell infusion. PET/CT revealed the EM leukemia recurred in multiple lesions, including lymph nodes in the mediastinum and right inner mammary region, intrapericardial, bilateral kidney, retroperitoneal, perirenal, and pelvic peritoneum ( Figure 1F ). Renal biopsy revealed the immunophenotype of leukemia cells was CD19+, with scattered CD20+/CD22+/CD34+/PAX-8+/CD79a+/TdT+/CD10+/CD3-/CD33-/CD99-, and CD1a- ( Figure 1J ;  Supplementary Figure S1 ). He achieved CRMRD- in the BM with transfusion-independent and EM leukemia burden marked decreased on PET/CT 1 month later ( Figure 1G ). Nonetheless, CD19+CD22+ blasts quickly rose to 96% in the BM along and EM leukemia recurred more than 1 month later ( Figure 1H ). Figure 1I ). Five months after CAR-T-cell infusion, the blasts count rose to 86.38% in the BM and were CD19-CD22+.
The structure of retroviral vectors encoding CARs. The CAR19-CD28-CD3zeta consisted of murine anti-CD19 scFv, a CD8 hinge region, CD28 transmembrane and cytoplasmic domain, and a CD3zeta cytoplasmic region. The CAR19-41BB-CD3zeta-mIL15 consisted of humanized anti-CD19 scFv, a CD8 hinge and transmembrane region, 4-1BB costimulatory domain, and a CD3zeta cytoplasmic region; mIL-15 is connected with CAR gene via a P2A peptide. The CAR22-41BB-CD3zeta-tEGFR consisted of humanized anti-CD22 scFv, a CD8 hinge and transmembrane region, 4-1BB costimulatory domain, and CD3zeta cytoplasmic region; tEGFR is integrated with CAR gene through a P2A peptide. mIL-15, membrane IL-15; EGFR, human epidermal growth factor receptor; tEGFR, truncated EGFR polypeptide; scFv, single-chain fragment variable.
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CARs design is shown in  Figure 2 .