DNA Damage in Haemopoeitic Stem Cells Impacts on Neutrophil and Platelet Engraftment Following Autologous Transplantation

Background and Aim: Processing and cryopreservation is known to reduce the viability and absolute number of CD34⁺ haematopoetic stem cells (HSCs). DNA damage and subsequent apoptosis of stem cells may occur as a result of prior treatment regimens or from the accumulation of reactive oxygen species (ROS) during the freeze/thawing process. In our recent study we demonstrated that two distinct populations of HSCs are present in all cryopreserved patient samples, based on their ROS expression; these we defined as ROS^high and ROS^low. The significant correlation between the percentage of ROS^high HSCs in the graft and neutrophil recovery following transplant shown in our study suggests that some degree of ROS accumulation may play an important role in promoting neutrophil engraftment following autologous HSC transplantation (AHSCT) (Bai et al 2018). The current study examined the association between ROS levels and DNA damage in cryopreserved HSCs and whether these correlated with time to neutrophil and platelet engraftment following AHSCT.

Methods: Cryopreserved HSC samples from 51 patients who underwent AHSCT were studied. HSC's were defined as CD45⁺/CD34⁺. DNA damage and intracellular ROS levels were assessed using an antibody against the phosphorylated form of histone H₂Ax (γH₂Ax) and 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA), respectively. Data acquisition and analysis were performed by flow cytometry.

Results: We observed a proportion of HSC in each sample that expressed elevated levels of gH2Ax. The median percentage of γH2Ax expressing CD34⁺ cells was 54.6% (range 3.4 - 98.4%). We observed a strong correlation between ROS^high and γH₂Ax levels in terms of the dose of cells/kg infused (p < 0.001, r = 0.72). The ratio of ROS^high/γH₂Ax-expressing HSCs was inversely associated with delayed neutrophil engraftment (p = 0.03, r = -0.31).

The cohort was divided into group 1 and group 2 based on ROS^high/γH₂Ax ratios of >1 (n = 32) and ≤1 (n = 18) respectively. Median days to neutrophil engraftment were 12 (range 9-18), in group 1 and 15 (range 9-28) in group 2, (p = 0.007). Median days to platelet engraftment were 17 (range 10-28) in group 1 and 21.5 (range 14-55) in group 2, (p = 0.001). Median days to platelet count of >50 (plt50) was 18.5 (range 12-28) in group 1 and 22 (range 16-75) in group 2, (p = 0.001). In group 2, significant correlations were noted between the ROS^high/γH₂Ax ratio and time to neutrophil engraftment (r = -0.54, p=0.025), plt20 (r = -0.64, p=0.004) and plt50 (r = -0.7, p = 0.002).

Conclusion: Our data suggest that DNA damage in CD34⁺ and accumulation of ROS may impact upon the time to engraftment of HSCs. Calculating a ratio of ROS^high/γH₂Ax expression may be useful in predicting engraftment time following autologous transplantation.