The first patient we present is a 2-year-old girl with an early, isolated bone marrow (BM) relapse of B-cell precursor (BCP)-ALL after allogeneic HSCT (Figure 1 and Table 1). The girl was diagnosed with an KMT2A-rearranged BCP-ALL (KMT2A-AFF1 gene fusion positive) at the age of 7 weeks. She received treatment according to the Interfant-2006 protocol for high-risk patients (Clinicaltrials.gov registration number: NCT00550992)   and underwent an allogeneic HSCT in first complete remission (CR1). At that time, she was negative for minimal residual disease (MRD) as measured by polymerase chain reaction (PCR) of immunoglobulin/T-cell receptor and KMT2A rearrangements. During front-line therapy, the patient had considerable treatment-related toxicity, including an episode of respiratory failure that required mechanical ventilation, repeated septicaemias and hepatopathy with pronounced hyperbilirubinaemia. After chemoconditioning with fludarabine, treosulfan, and thiotepa, the girl received a BM graft from a human leukocyte antigen (HLA)-matched unrelated donor, matched at 10 out of 10 loci. Graft-versus-host disease (GvHD) prophylaxis consisted of anti-thymocyte globulin, cyclosporine A and methotrexate. Further serious infectious complications occurred in the pre-engraftment phase, including an extensive soft tissue infection at the implantation site of her percutaneous endoscopic gastrostomy tube. This required broad-spectrum antibiotics and granulocyte transfusions. Neutrophil engraftment in the peripheral blood (PB) was documented on Day + 19. BM puncture on Day + 26 revealed molecular remission (MRD negative by both immunoglobulin/T-cell receptor- and KMT2A-based targets) with full donor chimerism.
The patient had a CD19+ (>99% CD19 expression), isolated BM relapse on Day + 180 after allogeneic HSCT. After having a multidisciplinary leukaemia and cellular therapy board session, we opted for tisagenlecleucel as remission induction therapy followed by allogeneic HSCT. Given the patient's body weight (10 kg), apheresis was performed via the AMICUS  system using allogeneic donor erythrocytes for machine priming. 148 x 106/kg CD3+ cells were harvested, resulting in a final product of 3.26 x 106/kg CAR T-cells. As bridging therapy prior to tisagenlecleucel, the patient received F1 and F2 courses according to the ALL-relapse study of the Berlin-Frankfurt-Munster Group (ALL-Relapse BFM 2002) (Clinicaltrials.gov registration number: NCT00114348)   with adjacent lymphodepletion (fludarabine, cyclophosphamide).
During bridging therapy, the patient had another soft tissue infection. Nevertheless, she remained MRD positive by PCR at a level of 7 x 10-1. Tisagenlecleucel was tolerated well; the patient did not develop cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Molecular remission in the BM was achieved on Day + 21. The patient proceeded to a second allogeneic HSCT using PB stem cells from her mother (who was HLA matched at 9 out of 10 loci) after fractionated total body irradiation (TBI) to a total dose of 8 Grey (Gy) and etoposide (60 mg/kg). GvHD prophylaxis consisted of post-transplant cyclophosphamide, tacrolimus, and mycophenolate mofetil. Toxicities of the second allogeneic HSCT included another soft tissue infection and mild veno-occlusive disease.
Engraftment in the PB was achieved on Day + 23. BM on Day + 32 showed complete donor chimerism with negative MRD by PCR. Immunosuppression was tapered and withdrawn on Day + 39 at complete absence of acute GvHD. Molecular remission and complete donor chimerism were confirmed on Day + 60, and Day + 100, and day +180.