Identifying Inherited and Acquired Genetic Factors Involved in Poor Stem Cell Mobilization and Donor-Derived Malignancy
Authors: Rojek Katarzyna , Nickels Eric , Niestadt Barbara , Marquez Rafael , Wickrema Amittha , Artz Andrew , van Besien Koen , Lee Ming K , Segal Jeremy P. , King Mary-Claire , Walsh Tom , Shimamura Akiko , Keel Sioban , Churpek Jane E , Godley Lucy A 
Published: October 2019

Abstract
Background
Mobilized peripheral blood (PB) stem cells (PBSC) are the most frequent source of hematopoietic stem cells (HSC) used for allogeneic HSC transplantation (HSCT). However, genetic factors contributing to donors who mobilize PBSC poorly are not well understood. We previously identified an undetected case of a familial myelodysplastic syndrome/acute leukemia (MDS/AL) predisposition syndrome in a donor with mild pre-donation thrombocytopenia who mobilized very poorly. Familial MDS/AL predisposition syndromes were identified in two other donors found to have hypocellular bone marrows after poor PBSC mobilization. Thus, we hypothesized that we could identify germline MDS/AL predisposition among poor mobilizing donors using a genomic screen of key hematopoietic genes.
Patients and Methods
Among 331 HLA-matched related HSC donors who underwent PBSC mobilization at The University of Chicago from 2001 to 2011, we defined those whose PB CD34+ cells/μl on day 5 post G-CSF administration fell within the lowest quartile as poor mobilizers (PM). Genomic DNA was available for 23 consented PM. This study was approved by The University of Chicago Institutional Review Board. Genomic DNAs isolated from donors' mobilized PBSC product were screened utilizing MarrowSeq, an next generation sequencing (NGS) assay targeting 142 genes involved in inherited and acquired bone marrow failure syndromes and hematologic malignancies. Potentially damaging variants were confirmed by Sanger sequencing. Clinical NGS panel testing was used to identify additional acquired mutations in one donor/recipient pair.
Results
Among the 23 PM sequenced using MarrowSeq, deleterious mutations were identified in 2 (9%). The first, a 63 year old male donor with mild thrombocytopenia (platelet count 136 K/μL) and macrocytosis (MCV 107.8 fL) was previously reported by our group to have a deleterious germline TERT mutation (c.2908A>G; p.M970V; 48% allelic ratio), which was also identified in the recipient's leukemia. This donor required bone marrow harvest after two failed PBSC mobilization attempts and the recipient died 5 months post-HSCT due to complications of poor engraftment. The second was a 67 year old mildly thrombocytopenic male donor (platelet count 139 K/μl) with a deleterious TET2 mutation (c.3765C>G; p.Y1255*; 48% allelic ratio), which was not present in the recipient's skin fibroblasts, most consistent with the presence of clonal hematopoiesis of indeterminate potential (CHIP) in the donor. Follow-up of this donor six years after collection of these TET2-mutated PBSCs demonstrated persistent mild thrombocytopenia (platelet count 121 K/μl), but otherwise normal blood counts. The recipient achieved remission after reinduction chemotherapy for relapsed AML followed by matched-related HSCT. Post-HSCT, the recipient demonstrated persistent mild thrombocytopenia. Six years later, she developed transfusion-dependent anemia and refractory anemia with excess blasts-2. Karyotype and microsatellite marker analysis of the bone marrow were consistent with an MDS of male donor origin. NGS sequencing from the bone marrow demonstrated the same TET2 mutation found in the mobilized PBSC product used in the transplant and an additional acquired RUNX1 mutation (c.585del; p.T196Qfs*15). Additional data from NGS sequencing of donor and recipient samples over time will be presented.
Conclusions
We demonstrate that both inherited and acquired genetic factors in PBSC from apparently healthy donors can contribute to poor mobilization and donor-derived malignancy post-HSCT. To the best of our knowledge, this is the first example of clonal evolution of TET2-mutated CHIP within a donor resulting in a donor-derived leukemia within the HSCT recipient. Replicative stress to reconstitute hematopoiesis in the recipient and/or the recipient's damaged bone marrow microenvironment may be contributing factors to the development of this donor-derived leukemia while the donor remains well. This study suggests that PM with thrombocytopenia may carry inherited or acquired mutations in hematopoietic genes, and alternative donor options should be considered. Our study also raises concerns about whether we need to screen for CHIP in healthy donors with unexplained cytopenias, especially as donors of increasing age are utilized.
This research was supported by the American Society of Hematology through the ASH HONORS Award.

Disclosures
van Besien: Miltenyi Biotec: Research Funding. Godley:Onconova: Research Funding.