Clonal Architecture of Donor-Derived Leukemia/Clonal Hematopoiesis after Allogeneic Hematopoietic Stem Cell Transplant

BACKGROUND Donor derived leukemia (DDL) is a rare complication of allogeneic hematopoietic stem cell transplant (alloHSCT) with reported incidence ranging from 0.08% to 0.84% and median time to diagnosis 17 to 48 months post-alloHSCT (Aldos et al. Leuk lymphoma 2021). Prognosis of DDL is dismal, with median overall survival (OS) of 5 to 11 months (Engel et al. Leukemia 2019). Here, we report the incidence of DDL/CH at our centre and explore the clonal characteristics and kinetics of expansion in DDL/CH alloHSCT recipients and respective donors. METHOD We retrospectively screened all South Australian (SA) alloHSCT recipients surviving ≥1-year post-alloHSCT for persistent unexplained cytopenia between 2015 and 2021, inclusive. Persistent cytopenia was defined as Hb <13g/dL in males and <12g/dL in females, absolute neutrophil count <1.8x109/L, and/or platelets <150x109/L. Recipients with relapse, extensive graft versus host disease (GVHD) requiring systemic immunosuppression or treatment related cytopenia were excluded. Additional cases of DDL/CH diagnosed clinically outside specified criteria were included for characterizing clonal architecture. Donor hematopoiesis was confirmed by chimerism assay via PCR amplification of short tandem repeats, with donor chimerism ≥95% accepted as evidence of complete donor chimerism. Serial patient and sibling donor samples (when available) were screened by Next-Generation Sequencing to evaluate clonal architecture and expansion. Serial Bone Marrow Mesenchymal Stromal Cells (BMSC) were assessed. RESULTS 282 patients had alloHSCT between 2015 and 2021 and 35.5% (n=100) were alive for ≥1 years, without relapse or severe GVHD requiring immunosuppression. Persistent unexplained cytopenia was observed in 9% (n=9) of these patients and 3 cases were confirmed to have DDL/CH. In order to characterize the architecture and kinetics, 12 additional cases of DDL/CH (total cases: n=15) were confirmed, identified in SA recipients transplanted outside the study period (n=9) or at other centres (n=3). Key findings include (i) unexplained persistent cytopenia observed in 9% of alloHSCT survivors without moderate/severe cGVHD; (ii) DNMT3Amut and TET2mut were most prevalent (67%; n=10/15) mutations in DDL/CH cases; (iii) 50% (n=4/8) of DNMT3Amut DDL/CH had single DNMT3Amut whilst TET2mut DDL/CH had either multiple TET2mut or other co-mutations (iv) DNMT3Amut DDL/CH had a shorter latency (median 21.5; IQR 3.74 - 42 months) as compared to TET2mut DDL/CH (median 299; IQR 1.48 - 343 months); (v) clonal kinetics of expansion varied between individual clones within the same alloHSCT recipient: in one case of DDL where persistent unexplained cytopenia developed 25 years post-alloHSCT, we observed TET2mut(p.Cys1378Tyr) variant allele frequency (VAF) progressively increase from 0% to 40% across those 25 years, whilst SRSF2mut (p.Pro95His) was not detected until 23 years post-alloHSCT when it presented abruptly with a significant VAF of 36%; (vi) kinetics of CH expansion varied immensely between donor and recipient: we saw a DNMT3Amut (p.Gly543Cys) VAF increase from 12.5% to 47% over 53 months post-alloHSCT in a recipient who developed persistent unexplained cytopenia 42 months post-alloHSCT and progressed to AML. However, the VAF of the same clone in the hematologically-stable donor was only 12.65% after 60 months of stem cell donation; (vii) DDL can be due to in vivo CH engrafted donor cells: one of our cases was diagnosed with myelodysplastic syndrome with three distinct TET2mut 29 years post-alloHSCT in the setting of complete door chimerism. Surprisingly, none of these clones were detected in the donor sample whilst a different TET2mut (p.Ser82LysfsTer2) was detected at VAF of 6%; (viii) BMSCs from DDL patients exhibit high level of senescence (52-83% β-galactosidase + cells), defective proliferative, and clonogenic potential, and aberrant differentiation capacity well before the diagnosis of DDL. CONCLUSION Persistent unexplained cytopenia was detected in 9% of long-term transplant survivors. We observed unique differences in kinetics of DNMT3Amut and TET2mut clones and identified two distinct mechanisms of DDL; CH can be inadvertently transferred during alloHSCT or can be developed in vivo in engrafted donor cells. Finally, highly senescent BMSCs secrete highly pro-inflammatory cytokines that are conducive for expansion of aberrant clones.