A 30-year-old male was referred to our hospital because of his refractory B-ALL. He underwent three cycles of induction chemotherapy (VTCLP: Vincristine; Tepirubicin; Cyclophosphamide; PEG-Asparaginase; Prednisone/VICP: Vincristine; Idarubicin; Cyclophosphamide; Prednisone/hyper CVAD B: Methotrexate; Cytarabine) but failed to achieve remission. On admission, he had severe pneumonitis. Bone marrow and peripheral blood smears identified the proliferation of lymphoblastic cells (87% of bone marrow nucleated cells), and karyotyping revealed an abnormal complex karyotype. NGS was performed and TP53 mutation was identified. After controlling his infection, he was recruited for a CAR-T clinical trial (Chictr.org N, ChiCTR-OPN-16008526) under informed consent in October 2017. The preparation of autologous CAR-T cells is described in detail in the  Supplementary Material . The patient received cyclophosphamide 300 mg/m2 and fludarabine 25 mg/m2 conditioning regimen on days -4 to -2 prior to CAR-T infusion. Anti-CD22 CAR-T cells (2 x 106/kg) and anti-CD19 CAR-T cells (2 x 106/kg) were infused on two consecutive days. He developed Grade 3 cytokine release syndrome (CRS). The minimal residual leukemia on day +28 after CAR-T infusion examined by FCM was negative. On April 13, 2018, the patient underwent allogeneic transplantation with granulocyte colony-stimulating factor mobilized bone marrow cells plus peripheral blood hematopoietic stem cells from his cousin, who was HLA-five loci mismatched. He received a TBI/VP/CY/ATG conditioning regimen consisting of total body irradiation (day -10), etoposide (15 mg/kg/day on days -8 and -7), cyclophosphamide (1.8 g/m2/day on days -6 and -5), and ATG (thymoglobulin, 2.0 mg/kg/day on days -4 to -1). Prophylaxis for GVHD consisted of cyclosporine and MMF (from day -1) and short-term methotrexate on days 1, 3, 6, and 11. The engraftment of neutrophils was achieved on day +11. Chimeric analysis showed the complete donor type on day 28, and no GVHD occurred. Quantitative EBV PCR monitoring was performed weekly. On day 34, measurement of the EBV-DNA level showed an increase to 143,000 copies/ml (from <500 copies/ml on day 27). A reduction in immunosuppression (the cessation of MMF, cyclophosphamide dose reduction) in combination with rituximab (375 mg/m2) weekly was initiated for the patient. However, his EBV-DNA copy numbers in PBMCs continued to increase, and subsequently, he developed fever and enlarged lymph nodes in the neck on day 50. A biopsy of the mass showed that the normal structure was destroyed, with diffuse invasion of cells showing atypical nuclear bodies, and immunostaining positivity for CD19, CD22, PAX5, MUM-1 and EBER; in addition approximately 80% of the tumor cells showed positive Ki-67 staining. However, CD20 was almost negative ( Figures 1A, B ). PET-CT detected hypermetabolic lesions and the involvement of the 86 cervical lymph nodes ( Figure 1C ). EBV-PTLD (CD20 negative) was diagnosed on the basis of the results. Given to the poor prognosis and the prominent presence of EBV, immunosuppression was completely discontinued, and CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) was given. However, his EBV viral load was still more than 103 copies/ml. Considering the insufficient number of T lymphocytes after CHOP therapy, donor T cells were collected for CAR-T preparation. Then, he received a total dose of 2 x 106/kg anti-CD22 and 2 x 106/kg anti-CD19 donor-derived CAR-T infusion from days 68 to 69. The timelines of EBV PCR and related treatments are depicted in  Figure 1E . On day 4 post-CAR-T infusion, the patient had grade 1 CRS presenting as fever and slightly elevated CRP/sIL6 ( Figure 1F ), resolved fully by symptomatic and supportive treatment. He developed acute GVHD (grade II) on day 78, and the administration of prednisolone at 1.0 mg/kg was initiated. Although grade 4 neutropenia and thrombocytopenia were prolonged for approximately 2 weeks ( Figure 1G ), the patient's cervical lymphadenopathy improved and responded well to CAR-T therapy. After recovery from myelosuppression, PET-CT showed a resolution of the cervical lymphadenopathy ( Figures 1D ), and a rapid reduction in the blood EBV load was observed (400 copies/ml). EBV-DNA could not be detected in the plasma during the follow-up.
Unfortunately, the patient continued to have multidrug-resistant pneumonia and elected to pursue hospice care, where he subsequently passed away on day 300 post HSCT.