Post-transplant transient abnormal myelopoiesis evolving from a GATA1 mutant clone in umbilical cord blood

doi:10.21203/rs.3.rs-4827968/v1

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Case Report

Post-transplant transient abnormal myelopoiesis evolving from a GATA1 mutant clone in umbilical cord blood

https://doi.org/10.21203/rs.3.rs-4827968/v1

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Transient abnormal myelopoiesis (TAM) generally affects newborns with Down syndrome and is associated with constitutional trisomy 21 and a somatic GATA1 mutation. Here we describe a case of TAM which evolved after umbilical cord blood transplantation (UCBT), whose origin was identified as a GATA1 mutation-harboring clone in umbilical cord blood (UCB) by detailed genetic analyses. A 58-year-old male who received UCBT for peripheral T-cell lymphoma presented progressive anemia and thrombocytopenia, and leukocytosis with blast cells in the peripheral blood (PB). Bone marrow (BM) aspiration showed granulocytic and megakaryocytic dysplasia with excess blasts whose karyotype was trisomy 21. Short tandem repeat analysis showed complete donor chimerism. He was initially diagnosed as donor-derived myelodysplastic syndrome (MDS) and treated with azacitidine, followed by secondary transplantation using unrelated BM, providing durable complete remission. Retrospective targeted-capture sequencing analysis of PB/BM samples collected at multiple timepoints identified trisomy 21 and a GATA1 mutation, suggestive of a diagnosis of donor cell-derived TAM (DC-TAM). Importantly, a minor clone with the same GATA1 mutation was detected in UCB by droplet digital PCR. DC-TAM is a rare UCBT-related complication which resembles MDS, but the identification of GATA1 mutation may be useful for its diagnosis. Our genetic analyses revealed that a pre-existing clone in UCB may contribute to the development of donor cell-derived hematologic neoplasms, highlighting the potential relevance of genetic screening of donor UCB.

Transient abnormal myelopoiesis

Donor cell-derived hematologic neoplasms

Umbilical cord blood transplantation

Down syndrome

Targeted-capture sequencing

Droplet digital PCR

Donor cell-derived hematologic neoplasms (DCHN) are rare but fatal complication following allogeneic hematopoietic stem cell transplantation (HSCT). Patients who develop DCHN have a very poor prognosis and usually require allogeneic HSCT from another donor [1]. Umbilical cord blood (UCB) has been widely used as an alternative stem cell source of allogeneic HSCT due to its rapid availability and human leukocyte antigen (HLA) tolerance. Although DCHN have a higher and earlier incidence after UCB transplantation (UCBT) compared to bone marrow transplantation (BMT) or peripheral blood (PB) stem cell transplantation [2], the details of the evolution of DCHN clones after UCBT have not been fully investigated.

Transient abnormal myelopoiesis (TAM), characterized by the proliferation of megakaryocytic blasts and/or dysplasia, affects approximately 10% of Down syndrome (DS) newborns and usually resolves spontaneously within a few months [3]. Here we describe a case of donor cell-derived TAM (DC-TAM) after UCBT, in which a shared GATA1 mutation between donor UCB and DC-TAM samples was identified using targeted-capture sequencing (targeted-seq) and droplet digital PCR (ddPCR) analyses.

A 58-year-old male underwent UCBT for refractory peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) from an HLA two-antigen mismatched unrelated male donor following a reduced-intensity conditioning regimen consisting of fludarabine (Flu), melphalan (Mel), and 4 Gy of total body irradiation (TBI). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine A (CsA) and methotrexate (MTX). He achieved neutrophil engraftment on day 20 after transplantation. On day 28, bone marrow (BM) aspiration showed hypocellular marrow that possessed 46, XY, and complete donor chimerism was confirmed by the analysis of BM mononuclear cells using a short tandem repeat (STR) analysis. On day 41, he developed grade III acute GVHD affecting the skin and gastrointestinal tract, which was treated with systemic prednisolone (PSL) in addition to CsA. After the improvement of acute GVHD, PSL and CsA were gradually tapered and discontinued on day 138.

On day 125, blast cells (3.0%) appeared in the PB, with mild anemia (hemoglobin concentration of 100 g/L) and thrombocytopenia (110×10⁹ /L), increasing to 15.5% by day 146. BM analysis on day 140 showed granulocytic and megakaryocytic dysplasia with excess blasts (6.0%) that possessed 47, XY, + 21 (Fig. 1a-b). The blasts were negative for myeloperoxidase, and positive for CD4, CD7, CD33, CD41, and CD61 based on flow cytometric analysis. STR analysis of BM cells showed complete donor chimerism. Therefore, he was diagnosed with donor cell-derived myelodysplastic syndrome with excess blasts 2 (DC-MDS-EB2) and treated with azacitidine at 75 mg/m² for five days from day 151. Then, blast cells disappeared in the PB on day 174. After receiving four cycles of azacitidine, he underwent allogeneic BMT from an unrelated male donor following a reduced-intensity conditioning regimen consisting of Flu, busulfan(Bu) and 4 Gy of TBI. Tacrolimus and MTX were used for GVHD prophylaxis. Both PTCL-NOS and DCHN remained in remission 65 months after the second transplantation. His clinical course is shown in Fig. 1c.

To identify somatic alterations involved in the development of DCHN in this case, we retrospectively performed targeted-seq of 376 genes implicated in hematologic malignancies [4] using BM (DCHN-BM) and PB (DCHN-PB) collected on day 140 and 150, respectively, after the onset of DCHN as well as recipient PB samples before UCBT (Recipient-PB) as a negative control. We identified GATA1 (c.C189A, p.Y63X) and KMT2C (c.G7270T, p.Q2424K) mutations in DCHN-BM and DCHN-PB samples (Fig. 2a, b). In addition, the copy number analysis using single nucleotide polymorphisms (SNP) detected trisomy 21 and confirmed complete donor chimerism in these samples (Fig. 2a, c). As GATA1 nonsense mutations are well known to cause TAM in DS neonates with trisomy 21 [5], this case was considered to be diagnosed as DC-TAM. To genetically dissect the evolution of DC-TAM clone, we performed targeted-seq of a donor UCB (Donor-UCB) sample and a BM sample collected on day 28 before the onset of DCHN (Pre-DCHN-BM), but found no alterations in these samples (Fig. 2a, b). Then, to detect the GATA1 mutation with higher sensitivity, we performed ddPCR analysis. We detected the GATA1 mutation in DCHN-BM (19.3%) and DCHN-PB (26.3%) samples, consistent with the targeted-seq. Remarkably, a small proportion of the GATA1 mutations were also detected in the Donor-UCB (1.0%) and Pre-DCHN-BM (3.5%) samples, while almost no GATA1 mutation was detected in the Recipient-PB (0.4%) sample used as a negative control (Fig. 2a, d). According to the documentation from the UCB bank, the donor was phenotypically normal at birth and a 6-month medical check-up. Taken together, these results suggest that a GATA1-mutated clone in UCB expanded and developed into DC-TAM after transplantation in this case.

TAM is morphologically and immunophenotypically indistinguishable from non-transient myeloid malignancies such as myelodysplastic syndrome, acute megakaryoblastic leukemia, and myeloid leukemia associated with DS [3]. Although this case was initially diagnosed as DC-MDS-EB2, genetic analysis revealed the presence of trisomy 21 and a GATA1 mutation, and the absence of additional driver mutations characteristic of myeloid malignancies, retrospectively supporting a diagnosis of DC-TAM. Given TAM resolves in a few months without treatment in more than 90% of patients [3], genomic profiling is important to differentiate TAM from other myeloid malignancies in cases who develop DCHN after UCBT.

Consistent with a previous report [6], DC-TAM developed four months after UCBT in this case. Thus, it is reasonable to suppose that the onset of DC-TAM is earlier than that of other DCHN [2]. These observations suggest that a minor TAM clone is already present in a UCB graft and DC-TAM develops without acquiring additional mutations. Indeed, we detected a GATA1 mutation with an extremely low allele frequency (< 1%) in the donor UCB cells using the ddPCR method. TAM generally develops through the addition of GATA1 mutations to constitutional trisomy 21 in DS neonates, but occurs even in phenotypically normal neonates with trisomy 21 mosaicism [3, 5]. Given the UCB donor was reported to be phenotypically normal, the UCB was likely to have trisomy 21 mosaicism with a minor TAM clone.

To our knowledge, there have been ten DCHN cases that showed the presence of shared mutations between donor and DCHN samples, suggestive of premalignant clones in donor cells, using next-generation sequencing (NGS) (Table 1) [7–15]. In these cases, somatic mutations associated with clonal hematopoiesis, such as DNMT3A, JAK2, and U2AF1, or germline mutations known as genetic predisposition for myeloid neoplasms, such as DDX41 and GATA2, were detected in donor cells before transplantation. Additional driver mutations, such as NPM1, FLT3, and NRAS, were detected only in DCHN samples, which suggests that these mutations were acquired after transplantation, resulting in the development of DCHN. Although the detection of a small proportion of preleukemic clone is technically difficult, genetic screening of myeloid driver genes, including GATA1, in donor grafts may be useful to prevent the development of DCHN after transplantation.

Author Contributions

M. S., K. S., K. K. conceived and designed the study. M. S., K. S., S. F., K. Y., J. K., and T. M. managed the patient. N. Y. and S. O. performed targeted-capture sequencing and droplet digital PCR analysis. Y. Kubota, M. S., Y. Kogure, K. M., and K. K. analyzed and interpreted the data. Y. Kubota, M. S., Y. Kogure, and K. K. wrote the initial draft. All authors reviewed and approved the final draft of the manuscript.

Acknowledgements

The supercomputing resources were provided by the Human Genome Center, the Institute of Medical Science, The University of Tokyo.

Funding

This work was supported by Japan Agency for Medical Research and Development (grant number JP23ek0109617).

Competing Interests

The authors have no relevant competing interests to declare.

Ethical approval

This study was approved by the ethics committees of Keio University School of Medicine (Tokyo, Japan) and Kyoto University (Kyoto, Japan) and conformed to the principles outlined in the Declaration of Helsinki for the use of human tissue or subjects. Informed consent was obtained from the patient.

Data availability

The data presented in this study are available from the corresponding author upon reasonable request.

Code availability

Custom code used in this paper is available upon request.

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Table 1 is available in the Supplementary Files section.

No competing interests reported.

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Post-transplant transient abnormal myelopoiesis evolving from a GATA1 mutant clone in umbilical cord blood

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Abstract

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Introduction

Case Presentation

Discussion

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Author Contributions

References

Table 1

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Supplementary Files

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