Clonal Hematopoiesis in Allogenic Stem Cell Transplant Survivors

Introduction: Clonal Hematopoiesis of Indeterminate potential (CHIP) is found in 10% of those over 60 years of age in the general population, and describes detectable somatic clonal mutations but with normal blood counts and indeterminate risk of clonal evolution. The genes involved are recurrently mutated in hematologic malignancies. Mutations associated with clonal hematopoiesis have been associated with a higher all-cause mortality and risk of death from cardiovascular disease as well as development of hematologic malignancies. Allogeneic stem cell transplant recipients reconstitute their hematopoiesis from a minimal dose of donor stem cells within a stressful marrow microenvironment. As such, there is scope for subclones to develop, with profound implications for late survivor health. Describing the origins and risks associated with CHIP in allogeneic stem cell transplant could affect treatment plans and provide new insight on complications affecting survivorship among this population. The objective of our study was to detect the frequency and types of myeloid mutations in long term allogeneic stem cell transplant survivors.

Methods: We performed a retrospective analysis of post-transplant cryopreserved peripheral blood mononuclear cell samples from 10 long term survivors who had previously undergone allogeneic stem transplant with myeloablative total body irradiation conditioning at the Hematology Branch, NHLBI, NIH. Samples were also available from seven of their matched donors. Samples were sorted by flow cytometry to identify T-cell (CD3), B-cell (CD19) and myeloid (CD14) lineages. Next Generation Sequencing [Neogenomics] was utilized to identify relevant single nucleotide variants (SNV) and insertion/deletions (indel) in 63 genes known to be mutated in myeloid malignancies. The genomic alterations within each of these genes were detected through proprietary bioinformatic analysis software, interpreted in conjunction with reference databases such as COSMIC and dbSNP, and verified by a board certified clinical molecular geneticist or molecular pathologist.

Results: The survivor population included 6 men and 4 women. Six patients were Hispanic, 3 were white and 1 patient was black. Age at transplant ranged from 18 to 68 years with a median of 36 years. Time from transplant ranged from 9.5 to 24.3 years with a median of 13.4 years. All had normal blood counts at the time of sample acquisition. A total of nine mutations were identified among four patients. The most common mutations were NOTCH 1 (4 mutations) and TET2 (2 mutations). Of the 4 patients with mutations, 2 had mutations deemed to be pathologic (DNMT3A [1]) or likely pathologic (TET2 [2]). When stratified per cell lineage, each cell line (CD19, CD3, and CD14) had 3 mutations. Of the 7 donors analyzed, 1 was found to have 2 mutations (TET2 [likely pathologic] and NOTCH1 [VUS]).

Discussion: Mutations when found, were concurrent in both myeloid and lymphoid lineages, suggesting against skewed T-cell proliferation. Mutations (6 total) were more common in the two survivors who were 60+ years of age, of which 1 mutation was deemed pathologic (DNMT3A) and 1 was likely pathologic (TET2). While this is higher than the rate expected in 60+ individuals in the general population, larger sample sizes are needed to determine true significance. Time from transplant between 5-10 years had a higher average number of mutations than either 10-15 or 15+ years. This observation is inconsistent with expected age-related development of clonal hematopoiesis. We speculate that this discordance could be related to greater hematopoietic stress in the early years post-transplant allowing for the accumulation of mutations followed later by an environment that selects for genomic stability. While a high portion of patients (40%) in our analysis were found to have mutations, the lower number mutations found beyond 10 years is a potentially encouraging result for transplant survivors. Combined with the observation that age 60+ at transplant correlated with a higher number of mutations, these results could indicate that stem cell transplants do not add much to the impact of age-related hematopoiesis as previously assumed. Donor age, which could not be addressed in this study, could also play a role. Suggested future studies include larger samples sizes that includes the association of CH with risk of CVD, GVHD, and other known complications with SCT.

Bender:Neogenomics: Current Employment. Monroy:Neogenomics: Current Employment. Battiwalla:Fate Therapeutics: Research Funding; JNJ/Janssen: Research Funding; Astra Zeneca/Gracell: Research Funding; Kite/Gilead: Research Funding.