ASXL1-Mutated Donor HSCs Evolved into FLT3-ITD Positive AML in the Unrelated Donor and FLT3-ITD Negative AML in the Recipient after Transplant
Authors: Liu Zhongwen , Liu Hongxing , Zhang Yanli , Kong Dai , Shi Jie , Zhang Yin , Wen Meiying , Song Yongping , Tong Chunrong , Chen Mingyi , Murphy William J. , Sun Kai 
Published: October 2019

Abstract
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 blood development, harbor a selective growth advantage compared to their non-mutated normal stem/progenitor counterparts, lead to the development of clonal hematopoiesis, and can subsequently gain additional mutations that confer full malignant potential. Here we report an unusual two cases of genetically distinct AML arising from a single pre-leukemic ASXL1 L866X mutant clone.
A 28-year-old female was diagnosed with acute B lymphoblastic leukemia by the classical morphology and immunophenotype. Cytogenetic evaluation showed a normal 46, XX, female karyotype. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) from a matched unrelated donor, a 33-year-old male, was performed. Donor peripheral blood derived HSCs were mobilized and administered to the recipient. On the day +50 after Allo-HSCT, cytogenetics showed a donor derived 46, XY, male karyotype. Consistent with this, chimerism analysis showed 100% donor chimerism . Twenty months after Allo-HSCT, she relapsed with AML with monocytic differentiation. Immunophenotyping revealed the blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD64, CD14 and MPO, and were negative for B-cell or T-cell markers. Cytogenetics showed a normal 46, XY, male donor-derived karyotype. Engraftment study continued to show complete donor chimerism in the bone marrow. Molecular study revealed the ASXL1 L866X mutation. Notably, the ASXL1 L866X mutation was not only found in DNA obtained from the recipient at the time of the original diagnosis of donor cell leukemia (AML), but also found in DNA obtained on day +50 after allo-HSCT when the recipient showed full donor engraftment as well as successful complete remission. Moreover, this ASXL1 L866X mutation was also observed in DNA from the healthy donor at the time of donation of peripheral HSCs. In contrast, this ASXL1 L866X mutation was not observed in DNA obtained from recipient specimens at diagnosis or during complete remission of acute B-lymphocytic leukemia before allo-HSCT. Therefore, these data confirmed that this ASXL1 L866X mutation originated from donor HSCs . Except for the ASXL1 L866X mutation, we did not find mutations in CEBPA, DNMT3A, ETV6 (TEL), FLT3-ITD, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PHF6, PTPN11, RUNX1, TET2, or TP53 genes in the recipient at diagnosis, remission, or relapse of donor cell leukemia.
Eighty-four months after donation peripheral HSCs, the previously healthy donor also developed AML. The blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD123, CD38, and MPO, and were negative for B- and T-cell markers. Cytogenetic analysis revealed 46, XY, male normal karyotype. The ASXL1 L866X and FLT-3/ITD mutation were identified in leukemia cells, but were not detected in his germline fingernail DNA. After four cycles of induction chemotherapy, he received HLA-identical sibling Allo-HSCT from his brother for salvaged therapy. He acquired complete donor chimerism on day +60 and FLT-3/ITD-positive cells disappeared. Two months later, the leukemia relapsed and the patient died from the disease.
The fact that 84 months after donation peripheral HSCs, the healthy donor,carrying the same ASXL1 mutation, was diagnosed AML with additional new gene mutation, FLT-3/ITD, was strongly supported the most recent theory that mutation acquisition occurs in functionally normal HSCs and clonal evolution of pre-leukemic HSCs precedes human AML. Moreover, our findings provide in vivo evidence to support a clonal expansion human model in which ASXL1 is an early initiation mutation while FLT3 is a subsequent driver mutation for abnormal HSCs clonal expansion and malignant transformation.

Disclosures
No relevant conflicts of interest to declare.