Pre-transplant MRD negativity predicts favorable outcomes of CAR-T therapy for pediatirc relapsed and Refractory Acute lymphoblastic leukemia followed by TCRαβ-depleted haplo-HSCT Free

Background and aim: TCRαβ-depleted HLA-haploidentical stem cell transplantation(TCD Haplo-HSCT) represents a promising curative option for children with relapsed and refractory acute lymphoblastic leukemia (R/R-ALL). MRD negativity before HSCT is associated with a significant benefit in terms of relapse. We developed a novel approach using CarT pre-HSCT and prophylactic haplo-Donor lymphocyte infusion (DLI) post-HSCT to decrease the relapse and improve transplant outcomes.

Patients and method: From December 1, 2020，to July 1, 2025, 65 Pediatric patients with acute lymphoblastic leukemia (median age 7 years; range 1-17 yeas) who underwent TCRαβ-depleted haplo-HSCT were enrolled. The male: female was 38:27. Twenty-two patients were in first complete remission (CR1), 33 in second CR(CR2), 10 in or beyond third CR(≥CR3). 55 patients received CAR-T treatment and achieved MRD-negative by flow cytometry before haplo-HSCT. 10 patients received chemotherapy or other therapy and only one patient was MRD-postive by flow cytometry before Haplo-HSCT. Fifty-one B-ALL patients received single CD19-CAR-T infusion, single CD22-CAR-T infusion, or CD19/CD22-CAR-T sequential infusion. Four T-ALL patients received CD7-CAR-T treatment. The median time from CAR-T treatment to haplo-HSCT was 43 days (range, 23-380 days). All children were given a fully myeloablative preparative regimen. The conditioning consisted of Cyclophosphamide+ Fludarabine+Thiotepa+Busulfan+Anti-human T lymphocyte immunoglobulin (ATG-F) or Anti-thymocyte globulin (ATG). TCRαβ-depleted grafts contained a median of 27.7 (range, 13.9-114.3)×10⁶CD34⁺cells/kg, 114.8(range, 34.9-323.6)×10⁶NK-cells/kg, and 35.1 (range, 11.2-142.4)×10⁶TCRγδ⁺T-cells/kg. No patient received any post-HSCT GVHD prophylaxis. Unmanipulated prophylactic haplo-DLI was administered monthly at an escalating dose of 1.0 (range, 0.3-2.7)×10⁵CD3⁺T-cells/kg , starting from a median of day +67 (range, 41-217）after transplantation.

Results: With a median follow-up of 29 (range, 0.5-53) months, the median time to neutrophil and platelet engraftment was 14 (range, 10–27) and 9 (range, 6–36) days, respectively. Five children experienced primary graft failure, four children were rescued after re-conditioning and a secondary haplo-HSCT. Final engraftment was achieved in 64/65 patients. Five patients died, the cumulative incidence of translate related mortality (TRM) was 7.9%, whereas five patients relapsed after transplantation (three patients who receiving donor-CAR-T therapy still alive), resulting in a 9.5% cumulative incidence of relapse. The overall survival (OS) and leukemia-free survival (LFS) was 90.1% and 83.2%, respectively. In comparing TCD haplo-HSCT in CR1、CR2 versus ≥CR3, the cumulative incidence of OS, LFS, Relapse and TRM at 2-years was 95.2%、93.9% vs.67.5% (P=0.063), 95.2%、83.2% vs. 58.3% (P=0.015), 0%、11.4% vs. 22.2% (P=0.031) and 4.8%、6.1% vs.21.2% (P=0.211). In comparingTCD haplo-HSCT with CAR-T versus TCD haplo-HSCT without CAR-T for the patients in or beyond CR2 (≥CR2) , the cumulative incidence of OS, LFS, Relapse and TRM at 2-yearswas 89.1% vs. 80% (P=1), 83.6% vs. 40% (P=0.09), 5% vs. 3.4% (P=0.747) and 6.2% vs.12.1% (P=0.365). Fifty patients received monthly escalating doses of haplo-DLI. Twenty-nine patients developed grade I-II aGVHD (25 cases in grade I, 4 cases in grade II), and one patient developed grade IV GVHD (skin-only). Seven patients developed cGVHD. The cumulative incidence of II-IV aGVHD and cGVHD was 9.1% and 12.7%, respectively.

Summary: TCD Haplo-HSCT can offer long-term remission for pediatirc R/R- ALL patients with minimal GVHD, including the patients who are beyond CR1 but achieve MRD-negative CR after CAR T-cell therapy.