Recently, it has been shown that hematopoietic stem cells (HSCs) acquire somatic mutations over time, most commonly involving DNMT3A, ASXL1, and TET2 genes . These mutation-bearing stem cells contribute to normal hematopoiesis harbor a selective growth advantage compared to their non-mutated normal stem/progenitor counterparts, leading to the development of clonal hematopoiesis, and can subsequently gain additional mutations that confer full malignant potential [1–3]. Although normal individuals with clonal hematopoiesis are at increased risk of developing hematologic malignancy, there are fewer studies to directly demonstrate pre-leukemic clone carrying a single epigenetic founder mutation can promote evolution to AML in vivo. In some patients, donor HSCs may contribute to the development of acute leukemia in the recipient. This is called donor cell leukemia (DCL) and almost always the leukemia is AML [4, 5]. Hahn et al.  reported that a pre-leukemic clone carrying a single epigenetic founder mutation (DNMT3AR882H) was transplanted from an apparently healthy marrow donor to his brother. After transplant, the sibling donor first developed a normal karyotype AML with mutations in DNMT3A, NPM1, and FLT3, and 4 months later, the recipient developed a normal karyotype AML with mutations in DNMT3A and NPM1, but wild-type FLT3 . This report presents direct evidence that HSCs carrying a single pre-leukemic mutation can evolve to AML .
Here, we report an unusual case. A female patient underwent successful, unrelated allogeneic hematopoietic stem cell transplantation (allo-HSCT) for B-ALL (Supplementary Fig. I). Twenty months after transplant of donor HSCs with a preexisting ASXL1-mutation, the recipient was diagnosed with FLT3-ITD-negative AML (Supplementary Fig. I). In addition, 84 months after transplant (64 months after the recipient was diagnosed with AML), the donor developed FLT3-ITD-positive AML carrying the same ASXL1-mutation (Supplementary Fig. I). Before allo-HSCT, the recipient and the donor gave their written informed consent for “potential usage of specimens for medical research” in accordance with the Declaration of Helsinki.
The 28-year-old female was diagnosed with B-ALL by classical morphology and immunophenotype (Supplementary Fig. I). Cytogenetic evaluation showed a normal 46, XX. The patient achieved CR with induction therapy. After consolidation therapy, an allo-HSCT from an HLA-identical unrelated donor (33-year-old male, donor-recipient matched by high resolution HLA typing at HLA-A, -B, -C, DRB1, and DQB1, 10/10 matches) was performed (Supplementary Table 1). The recipient received conditioning with busulfan, 4 mg/kg/day orally for 3 days; cyclophosphamide, 50 mg/kg/day for 2 days; cytarabine, 2 g/m2/day for 1 day; semustine, 250 mg/m2/day orally for 1 day; and rabbit anti-human thymocyte immunoglobulin (rATG, Thymoglobuline, Genzyme), 1.8 mg/kg/day for 4 days. Donor peripheral blood stem cells (PBSC) were mobilized, pheresed and administered to the recipient. GVHD prophylaxis consisted of traditional cyclosporine, short-course methotrexate (15 mg/m2 at day +1, 10 mg/m2 at days +3, +6, and +11) and mycophenolate mofetil 0.75 g, orally, twice daily for 1 month. On day +50 after allo-HSCT, cytogenetics showed a donor-derived 46, XY. Consistent with this, microsatellites and capillary electrophoresis-based chimerism analysis showed 100% donor chimerism (Fig. 1). However, 20 months after allo-HSCT, the patient again appeared to have symptoms of hematopoietic abnormalities and was diagnosed with monocytic differentiation AML with 45.2% monoblasts in the bone marrow. Immunophenotyping revealed that the blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD64, CD14, and myeloperoxidase (MPO) and were negative for B-cell or T-cell markers (Supplementary Fig. I). Cytogenetic analysis showed a normal 46, XY, donor-derived karyotype. An engraftment study continued to show complete donor chimerism in the bone marrow (Fig. 1). Mutation screening  for 11 genes including ASXL1, CEBPA, DNMT3A, FLT3-ITD/TKD, IDH1, IDH2, KIT, NPM1, PHF6, TET2, and TP53 by Sanger sequencing showed an ASXL1 c.2597T>A/p.L866X mutation (Fig. 2), and the allelic burden was identified as 43.6% by clone sequencing (Supplementary Table 2). After the induction therapy of AML, the recipient achieved CR, which was sustained for 7 months by regular consolidation chemotherapy. Then, AML relapse occurred. The morphology, immunophenotype, and cytogenetics of the leukemic cells remained the same as the original AML diagnosis. The patient did not achieve CR and finally died of severe infection 4 months later.
Eighty-four months after donation of PBSC, the previously healthy donor also developed AML with 30.5% myeloblasts in the bone marrow. The blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD123, and MPO and were negative for B- and T-cell markers. Cytogenetic analysis revealed 46, XY. Mutation screening  for ten genes showed an ASXL1 c.2597T>A/p.L866X and FLT3-ITD (FLT3 c.1780_1781delinsCAGGTGGCACAGGTTCCACTCCC/p.F594delinsQVAQVPLP) mutation. The mutant allelic burdens of ASXL1 and FLT3-ITD were 38.5% and 42.2%, respectively. After four cycles of induction chemotherapy, he received HLA-identical sibling allo-HSCT from his brother for salvage therapy. He acquired complete donor chimerism on day +60, and FLT3-ITD-positive cells disappeared. Two months later, the leukemia relapsed, and the patient died from the disease.
We then retrospectively performed mutation screening in the obtained samples (Supplementary Table 2). An ASXL1 c.2597T>A mutation was detected in PBSC from the donor at the time of donation and in bone marrow cells from the recipient on day +50 after allo-HSCT, with an allelic burden of 29.7% and 36.1%, respectively (Fig. 2, Supplementary Table 2). FLT3-ITD mutation was negative in the obtained recipient samples, in PBSC from the donor at the time of donation and in a fingernail specimen from the donor after the diagnosis of AML by sanger sequencing (Supplementary Table 2). We further designed sequence specific primers (forward 5’-AGCAATTTAGGTATGAAAGCCAGC-3’FAM, reverse 5’-GAACCTGTGCCACCTGATCAA-3’) for these samples to detect the FLT3-ITD mutation with a sensitivity of 0.01%, but we still found negative results.
In the case described here, an ASXL1-mutation was not only found in DNA from the recipient’s specimens taken at the time of the original diagnosis of the recipient’s donor origin AML but also found in DNA from the recipient’s specimens taken at the time of the early donor cell engraftment with full-donor chimerism after allo-HSCT, which is over 18 months before DCL was diagnosed. This ASXL1 c.2597T>A mutation was also observed in DNA from the healthy donor at the time of donation of the PBSC. In contrast, the same ASXL1-mutation was not detected in DNA from the recipient’s specimens taken at the time of the original diagnosis of ALL and during the remission before allo-HSCT. Thus, the ASXL1 L866X mutation was present in donor HSCs before transplantation.
Except for the ASXL1 L866X mutation, we did not find mutations in CEBPA, DNMT3A, FLT3-ITD/TKD, IDH1, IDH2, KIT, NPM1, PHF6, TET2, and TP53 genes in the recipient at diagnosis, or at relapse of DCL. Thus, both the recipient’s new AML and the donor’s AML were presumably derived from donor HSCs with a preexisting ASXL1-mutation. Although we have no direct evidence to show additional mutations in the recipient with DCL, the fact that after donation of PBSC, the healthy donor was diagnosed with AML with an additional new gene mutation, FLT3-ITD, strongly supports the most recent theory that mutation acquisition occurs in functionally normal HSCs and that clonal evolution of pre-leukemic HSCs precedes human AML [8, 9]. Moreover, our findings provide in vivo evidence to support a clonal expansion in a human model in which ASXL1 is an early initiation mutation, while FLT3 is a subsequent driver mutation for abnormal HSC clonal expansion and malignant transformation [8, 10]. In accord with our case, Hahn et al.  also identified a detectable FLT3 mutation in the donor with clonal DNMT3A-mutant HSCs at the time of AML diagnosis, which would be helpful in understanding possible trends in leukemogenesis. It is rather remarkable that in both donors, different early initiation mutations might be associated with different latency times of leukemogenesis. In our case with an ASXL1 mutated clone as the initiation mutation, the leukemia was much slower to develop in the donor and very different from the disease that had developed in the recipient. In their case, the DNMT3A-mutated clone progressed to AML at similar times in donor and recipient, which may argue against the hypothesis that DCL arises faster in the recipient due to increased demand for proliferation [6, 11]. In summary, we report on two unique, sequential patients with genetically distinct AML presumably arising from donor HSCs with a preexisting ASXL1-mutation that are instructive for studying in vivo leukemogenesis. One of the clinically important questions arising from this case regards the need for pre-leukemic mutation screening as part of the sibling or haploidentical-related donor selection and tracking. It is still premature to extrapolate from this case to the conclusion that unrelated donors should be routinely screened for the presence of clonal hematopoiesis before transplantation, as very few cases of overt AML have been identified in unrelated donors following the development of DCL in the recipient. However, additional cases of DCL should be studied to see if the donor cells in such cases harbor mutations in genes that may be involved in leukemogenesis.
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We thank Dr. Dan L. Longo (Deputy Editor, New England Journal of Medicine) for invaluable guidance in the preparation of the manuscript. This study was partially supported by the National Natural Science Foundation of China (No. 81273259, No. 81471589), the Health Bureau of Henan Province, P.R. China (No. 201201005) and the foundation and frontier research grant of Henan provincial science and technology bureau, P.R. China (No.112300410027, No.142300410078).
ZL performed the clinical research and contributed vital clinical data. HL and FW designed the research, performed research, and contributed analyzed data. YZ performed the clinical research and contributed vital clinical data. DK, MS, HAWA, LH performed clinical research and contributed vital clinical data. MW performed research and contributed vital samples of donated PBSC. MC, WJM, and KS designed the research, analyzed the data, and wrote the paper.
Conflict of interest
The authors declare that they have no conflict of interest.