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.

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2025
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