Double Umbilical Cord Blood Transplantation Preceded by a Reduced-Intensity Conditioning Regimen: Rapid Induction of Single Donor Chimerism and Highly Predictive Value of Early CD4+ T Cell and NK Cell Predominance

Double umbilical cord blood transplantation (UCBT) results in higher engraftment rates as compared to single UCBT in adult patients. Sustained hematopoiesis is usually derived from a single cord blood unit (CBU) after double UCBT. So far, the mechanism of predominance of a particular CBU is unresolved. In a prospective single-arm phase II study (HOVON-106) we monitored early engraftment kinetics to determine whether graft predominance after double UCBT is driven by specific leukocyte subpopulations. Methods 36 consecutive patients (pts) from 5 Dutch centers with high-risk hematological diseases received a double UCBT, preceded by a reduced-intensity conditioning regimen (Cy 60 mg/kg/ Flu 160 mg/m²/ TBI 2×2 Gy). CBUs were selected by intermediate resolution typing for HLA-A and -B loci and by high-resolution typing for HLA-DRB1. The minimal required HLA-match grade was 4/6. Chimerism analysis (STR-PCR) of unseparated peripheral blood (PB) and bone marrow (BM) cells was performed as from day +32 onwards. In addition, chimerism analysis in PB leukocyte subpopulations by flowcytometry using lineage-specific (CD45, CD3, CD4, CD8, CD19, CD16/56, CD14 and CD33) monoclonal antibodies (mAbs) in combination with human HLA-antigen specific mAbs (HLA-mAbs) was performed at day +11, +18, +25 and +32 if discriminating HLA-mismatches between recipient and CBUs were present. Day +32 flowcytometry results were compared to day +32 PB STR-PCR results. Results The median number of prefreeze total nucleated cells (TNC) per CBU was 2.3×10⁷/kg (range: 1.5–5.5). Median numbers of post-thaw viable CD34+ cells, T-, B- and NK cells were 0.32 (range: 0–1.7), 4.4 (range: 0.4–36), 9.7 (range: 0.7–79) and 7.2 (range: 0.24–45) x10⁵/kg, respectively. One pt was non-evaluable for engraftment due to insufficient follow up after early relapse. The cumulative incidence of neutrophil recovery (≥0.5×10⁹/l) was 91% with a median time to neutrophil recovery of 33 days (range: 15–82). Primary graft failure occurred in 1 pt. Chimerism analysis, performed at day +32 by STR-PCR revealed single CBU predominance in all pts whereas residual non-engrafting CBU and recipient cells were detectable in only 3 and 7 pts, respectively. Simultaneous 3-donor-origin detection of leukocyte subpopulations by flowcytometry based on HLA disparities was possible in 12 pts. Flowcytometry using HLA-mAbs demonstrated single CBU predominance in various leukocyte subsets as from day +11 onwards in the majority of pts. Moreover, ultimate engraftment of a particular CBU was reliably predicted for by chimerism within the CD4+ (in 90% of pts) and NK cell (in 90% of pts) subsets at this early time point. In contrast, predominance of the engrafting CBU in monocytic en myeloid subsets was observed in only 70% and 33% of pts, respectively, at day +11. The numbers of CD8+ and B-cells were too low for analysis in the majority of pts. Predominance of the ultimate engrafting CBU was established in all subpopulations at day +18. Furthermore, the contribution of the non-engrafting CBU to the different leukocyte subsets was negligible or even absent as from day +18 onwards. Recipient hematopoiesis did not contribute to PB cell recovery either. The results of day +32 flowcytometry (CD45+ population) were similar to results of day +32 PB STR-PCR. The number of prefreeze TNC did not predict for the ultimately engrafting CBU, nor did the number of post-thaw CD34+, T, B or NK cells. Engraftment was not associated with the degree of HLA mismatches or presence of KIR ligand mismatches among recipient and CBUs. Conclusions These results show that single donor chimerism is rapidly established after double UCBT, preceded by a 4 Gy TBI-based conditioning regimen without ATG. In addition, our flowcytometry data suggest the occurrence of CBU predominance within 2 weeks post transplant, in the course of which both CD4+ and NK cell predominance at day +11 are highly predictive for ultimate single donor chimerism. That early engraftment pattern of leukocyte subsets might suggest a key role for either CD4+ T cells or NK cells in CBU predominance.

Janssen:Novartis: Consultancy.