Persistence of the Losing Unit Following Double Umbilical Cord Blood Transplant: Finding the Unseen

Background: After double cord blood transplantation (dCBT), long-term hematopoietic dominance of a single cord blood (CB) donor graft is established in the majority of patients. As measured by standard clinical testing for chimerism (usually by single tandem repeat polymorphism STR), the "losing" unit usually becomes undetectable within the first month after transplantation. However, anecdotal cases in which the losing unit reemerges and contributes to hematopoiesis suggest long-term persistence of the losing unit in a quiescent state. In this study, we sought to determine whether, in the clinical setting of complete single CB unit dominance after dCBT, detection of very low levels (microchimerism, "Mc") of the losing unit could be demonstrated using a sensitive technique developed for microchimerism analysis.

Methods: We studied 15 patients who underwent myeloablative dCBT with either high dose TBI (13.2 Gy) (n=8) or treosulfan based conditioning regimens (n=7). Patients were included if an HLA polymorphism unique to the losing CB unit could be identified and targeted using a panel of HLA-specific quantitative polymerase chain reaction (QPCR) assays developed for microchimerism testing (sensitivity approximately 1 cell per 20,000). Of the 15 subjects, 14 were selected to demonstrate single CB unit dominance by clinical STR testing.  Samples were tested blinded to clinical information and one subject had been included with stable mixed unit-unit chimerism. We studied bone marrow (BM) and peripheral blood (PB) samples on days 28, 80, and 1 year after CB transplantation. From PB, cell lineage subsets (CD3+, CD33+, and CD56+) were sorted by fluorescence-activated cell sorting at the time of the blood draw. For each subject, DNA was extracted from bone marrow and cell subsets, and detection of the losing unit was evaluated using QPCR targeting the non-shared HLA polymorphism unique to the losing unit. Results are expressed as detection or not of the losing unit, as well as quantitative measurement of the concentration expressed as genome equivalents (GEq) of DNA from the losing unit per total GEq of DNA tested.

Results: The median age was 35.7 years (range, 10.1-61.7) and weight 72.2 kg (range, 32.5-107). The median follow-up was 1,163 days (range,138-2269). Disease status pre-transplant included acute myeloid leukemia (n=10), acute lymphoblastic leukemia (n=3), myelodysplastic syndrome (n=1), and myeloproliferative disorder (n=1). No patients experienced primary graft failure. For the overall study population (n=15), the median time to engraftment was 22.5 days (range 14-44). Of the 14 subjects with clinical single unit dominance, absence of the losing unit by standard clinical STR testing was confirmed at a median time of 20.1 days (range 11-28). The subject with clinical mixed-unit chimerism that had been included in the blinded testing gave positive results as expected. QPCR testing results are shown in Table 1. Overall, QPCR testing showed detection of the losing unit in 11/14 (79%) subjects. In the CD3 subset 4/13 (31%), 7/13 (54%), and 3/12 (25%) of evaluable patients showed detection of the losing unit at days 28, 80, and 365, respectively. In the CD33 subset, 6/12 (50%), 4/14 (29%), and 2/11 (18%) of evaluable patients showed detection of the losing unit at days 28, 80, and 365, respectively. In the CD56 subset, 4/13 (31%), 2/14 (14%), and 3/11 (27%) evaluable patients showed detection of the losing unit at days 28, 80, and 365, respectively. In the BM samples, 6/14 (43%), 1/14 (7%), and 1/10 (10%) of evaluable patients showed detection of the losing unit at days 28, 80, and 365, respectively. In the sorted cell subset samples, the median amount of GEq tested was 3.4x10³ (range 2.5x10² - 5.0x10⁴). In the BM samples, the median amount of GEq tested was 1.0x10⁵ (range 1.5x10⁴ - 1.7x10⁵).

Conclusions: Using a highly sensitive panel of polymorphism-specific QPCR assays, we have demonstrated persistent detection of the losing CB unit after dCBT, despite single unit dominance by clinically standard STR-based testing. Demonstration of losing unit reemergence in each subset suggests a dynamic phenomenon. We are currently extending the numbers of study subjects to determine whether the pattern of detection of the losing unit correlates with clinical outcomes after dCBT.