Early Mixed Lymphoid Chimerism in Acute Myeloid Leukemia Patients Undergoing Allogeneic Transplantation after a Myeloablative Busulfan-Based Conditioning Regimen
Authors: Cavallaro Gianluca , Pavoni Chiara , Algarotti Alessandra , Micò Maria Caterina , Grassi Anna , Lussana Federico , Finazzi Maria Chiara , Rizzuto Giuliana , Rambaldi Benedetta , Salvi Anna , Spinelli Orietta , Rambaldi Alessandro 
Published: December 2024

Introduction
The combination of fludarabine with a myeloablative dose of busulfan has proven to lower post-transplant non-relapse mortality (NRM) with regards to BuCy in a phase 3 clinical trial, with no detrimental effect on relapse incidence (CIR) (Rambaldi A., et al, Lancet Oncology, 2015). However, in this study a higher incidence of mixed lymphoid chimerism was observed in the BuFlu arm in the early post-transplant period. This analysis aims to define the frequency and clinical impact of early mixed lymphoid chimerism in patients with acute myeloid leukemia (AML) undergoing allogeneic stem cell transplantation (alloHSCT) with a myeloablative busulfan-based conditioning regimen.
Methods
We retrospectively analyzed 217 patients undergoing alloHSCT for AML at our Institution from 2008 to 2023. The study population was divided into two groups: patients receiving BuFlu (n=140) and those receiving a double alkylating (DA) conditioning regimen, either busulfan-cyclophosphamide or busulfan-thiotepa-fludarabine (n=77). For this analysis, we only considered patients reaching at least the 30-day chimerism evaluation. The analysis of clinical outcomes (cumulative incidence of relapse [CIR], Non-Relapse Mortality [NRM], incidence of acute and chronic graft-versus-host [aGvHD, cGVHD], and graft-and relapse-free survival [GRFS]) was performed in 136 patients treated with BuFlu arm and 71 in DA arm; patients who died within 30 days (4 in BuFlu group, 6 in DA group) were excluded. Hematopoietic chimerism was molecularly evaluated by Variable Number of Tandem Repeats (VNTR) on bone marrow (BM) mononuclear cells or peripheral blood (PB) T lymphocytes, purified by immunomagnetic positive selection (Clinimacs, Miltenyi, Biotec). The analysis was performed at days +30, +60, +90, +180, and +365 after alloHSCT, along with immune reconstitution of T lymphocytes subclasses (total CD3+, CD4+, CD8+ lymphocytes), B lymphocytes, and NK cells. Mixed chimerism was defined as less than 95% of donor cells in total BM mononuclear cells or the peripheral blood T lymphocytes.
Results
The median follow-up of the whole study population was 3.2 years (range 0.1-15.5). We confirmed a higher incidence of mixed lymphoid chimerism in patients treated with BuFlu (60.3% vs 18.3%, respectively, p&lt; 0.001), that remained significant up to 180 days after transplant: 56.5% vs 14.3% at day +30 (p&lt; 0.001); 36% vs 9.1% at day +60 (p 0.001); 26.4% vs 5% at day +90 (p 0.006); 12.7% vs 1.8% at day +180 (p 0.0201). Of the patients treated with BuFlu and showing a mixed lymphoid chimerism at day +30, 48% converted to full donor chimerism at day +60, 63% at day +90, and 84% at day +180. In 15 patients a donor lymphocyte infusion (DLI) was decided for chimerism induction (13 in BuFlu, 2 in DA group). Among the 11 patients who received DLI in the BuFlu group before day +100, 8 converted to full donor chimerism by day +365. Patients with PB lymphoid chimerism &lt; 75% by day +90 had a higher probability of receiving a DLI, as compared to those with a 75%-95% chimerism (p&lt; 0.0001). A ROC analysis showed that 72% is the best lymphoid chimerism threshold at day +30 discerning the possibility of spontaneous conversion to full donor chimerism by day +90. Patients with mixed lymphoid chimerism had delayed T-cell reconstitution, no matter the conditioning regimen or the GvHD prophylaxis. Univariate analysis showed that mixed lymphoid chimerism was associated with the BuFlu conditioning but not the stem cell source, the GvHD prophylaxis (post-transplant cyclophosphamide vs anti-T-lymphocyte globulin), and the number of CD3+ lymphocytes infused with the stem cell graft. The higher incidence of mixed lymphoid chimerism did not translate into an increased CIR, no matter the proportion of lymphoid chimerism. By univariate analysis, aGvHD as well as GRFS, NRM, and OS were not associated with mixed lymphoid chimerism. However, aGvHD was less frequent in patients receiving BuFlu vs DA (46% vs 26%, p 0.0142).
Conclusions
An early PB mixed lymphoid chimerism is frequent in BuFlu recipients and in most cases the conversion to full donor chimerism is progressive and does not require DLI. Early mixed lymphoid chimerism does not adversely affect the main clinical outcomes, of AML patients receiving a myeloablative BuFlu conditioning regimen.