Prognostic impact of mixed chimerism with undetectable measurable residual disease in pediatric and adolescent young adult patients undergoing allogeneic hematopoietic stem cell transplantation for hematological malignancies

Abstract Background Mixed chimerism (MC), defined as <95% donor chimerism post-allogeneic HSCT, has traditionally been associated with increased relapse risk in hematologic malignancies. However, its prognostic significance in patients with undetectable minimal residual disease (uMRD) remains uncertain. Emerging data suggests a potential protective role of donor-derived T and NK lymphocytes in sustaining remission despite MC. This study aims to assess the incidence, clinical impact, and immunologic significance of MC with uMRD, with a focus on lineage-specific chimerism (particularly T and NK cells) and correlations with infused CD34+ and T lymphocyte doses. Methods We conducted a multicenter retrospective study of 290 pediatric and adolescent and young adult (AYA) patients (median age 9.5 years; range 0.5–21) who underwent HSCT between January 2015 and December 2024 across 4 Italian centers. All patients had hematologic malignancies (ALL, AML, MDS, or lymphoma) and were in complete remission with uMRD at transplant. Chimerism was monitored at days +28, +60, +90, then quarterly for 2 years, and annually thereafter. Lineage-specific chimerism (myeloid, B, T, and NK lymphocytes) was analyzed using short tandem repeats (STR) after cell sorting (BD FACS Melody™). Results Overall survival and relapse-free survival at last follow up were 77% and 82%, respectively. MC with uMRD occurred in 9 patients (3%) and was not associated with relapse. Median age at transplant in this subgroup was 5.3 (0.5–12.7) years. Diagnoses included ALL (n=4), AML (n=3), MDS (n=1), and NHL (n=1). Donors were matched unrelated (n=7), haploidentical (n=1), and HLA-identical sibling (n=1). Conditioning regimens included TBI-based (n=4), busulfan-based (n=2), and treosulfan-based (n=2). Stem cell sources were bone marrow (n=7) and peripheral blood (n=2). GVHD prophylaxis included ATG (n=8), PTcy (n=1), and CSA combined with MTX or MMF (n=9). Median time to first MC detection was 17 (1–124) months post-HSCT, with a median MC duration of 26 (9–157) months. MC development was independent of demographic or clinical variables, including ongoing immunosuppression. All cases, but 2 showed high donor-derived T (>90%), with 3 patients achieving full (100%) T-cell chimerism. NK chimerism was 40, 60 and 80% in 3 tested patients, supporting a possible role in post-transplant immunosurveillance. Despite correlation between infused T lymphocytes and T-cell chimerism (p=0.368), as well as CD34+ cell doses and T-cell chimerism (p=0.757), were not statistically significant, observed linear trends support further investigation. Infectious complications included CMV (n=4), EBV reactivation (n=2), and one case of bacterial pneumonia. Acute GVHD (≥ grade II) occurred in 2 patients; chronic GVHD in 2; all cases responded to steroid therapy. All MC patients remained in sustained complete remission with uMRD at a median follow-up of 36 months. Conclusions In pediatric and AYA patients, MC with uMRD post-HSCT does not predict relapse. Sustained high donor chimerism in T and NK compartments may underlie durable remission. Lineage-specific chimerism analysis offers superior prognostic value over global assessments. Standardized monitoring protocols are essential to guide post-HSCT management and avoid unnecessary and harmful interventions.