Acute basophilic leukemia associated with the t(16;21)(p11;q22)/FUS‐ERG fusion gene

Key Clinical Message We herein report a rare case of acute basophilic leukemia with t(16;21)(p11;q22) generating the FUS‐ERG fusion gene. The basophilic nature of leukemia blasts was demonstrated by cytomorphology, toluidine blue metachromasia, mature basophil‐associated antigen expression, and characteristic granules under electron microscopy. The molecular link between t(16;21)/FUS‐ERG and basophilic differentiation remains unclear.


Introduction
Acute basophilic leukemia (ABL) is listed as a separate entity in the acute myeloid leukemia (AML), not otherwise specified category in the 2008 WHO classification, comprising <1% of all cases of AML [1]. The very recently proposed criteria of ABL are blasts ≥20% and immature basophils ≥40% of nucleated bone marrow (BM) or peripheral blood (PB) cells [2]. However, as the disease is defined on the basis of morphology, limited information is currently available on specific cytogenetic and molecular markers.
The t(16;21)(p11;q22) translocation was initially described in a patient with AML characterized by a large number of abnormal eosinophils in BM [3]. Sixty-six cases carrying t (16;21) are currently listed in the Mitelman database, which shows that this translocation is present in all ages and is associated with poor survival [4]. In 1994, two independent groups found that t (16;21) led to the generation of a fusion gene between FUS (FUS RNA binding protein) at 16p11 and ERG (ERG, ETS transcription factor) at 21q22, encoding the FUS-ERG chimeric protein [5,6]. The leukemogenic activity of this chimeric protein was demonstrated by the retroviral transduction of FUS-ERG to human umbilical cord blood cells, showing altered myeloid and arrested erythroid differentiation and marked increases in the proliferative and self-renewal capacities of transduced myeloid progenitors [7].
We herein report a rare case of ABL carrying the t (16;21)(p11;q22)/FUS-ERG fusion gene. Cytomorphological, electron microscopic, and cytogenetic features as well as the treatment course are described.

Case Report
A 65-year-old man was admitted to the Hematology Department of our hospital with a diagnosis of acute leukemia. Two weeks before, he developed swelling in the right lower leg that extended to the ankle and foot. He presented to the Orthopedic Department, and a blood examination revealed marked leukocytosis. There was no surface lymphadenopathy or splenomegaly. He did not have any symptoms suggestive of the excess release of histamine.
Bone marrow showed 30~40% cellularity containing 95.0% blasts. Leukemia blasts in PB and BM were of medium size and had round or indented nuclei with dispersed chromatin. Nucleoli were inconspicuous. Approximately, 60% of PB and 70% of BM blasts had variable amounts of a light-blue cytoplasm containing coarse basophilic granules ( Fig. 1A-C), and occasional cells showed a mature basophil appearance (Fig. 1D). The granular content often appeared to be extracted to generate cytoplasmic vacuoles (Fig. 1E). No myelodysplastic or hemophagocytic pictures were found. Although 31% PB and 9% BM blasts were positive for myeloperoxidase (MPO), positivity was weak (Fig. 1F). Cytoplasmic granules exhibited metachromasia with toluidine blue staining in 49% PB and 47% BM blasts (Fig. 1G). Chloroacetate esterase and a naphthyl butylate esterase were negative.

Differential Diagnosis and Investigations
Flow cytometry (FCM) revealed that leukemia blasts showed an immature myeloid cell phenotype, that is CD13 + , CD33 + , CD34 + , and CD117 + , but lacked HLA-DR expression ( Fig. 2A). Approximately, 20% of cells expressed CD203c, which was previously reported to be specific for basophils in PB and increased in response to IgE-dependent cell activation [2], and the acquisition of the antigen appeared to correlate with the loss of CD34 (Fig. 2B). Other antigens expressed were CD7 À , CD11b dim , CD25 + , CD45RA dim , CD45RO + , CD56 + , CD66c À , and CD123 + . Electron microscopy showed that leukemia blasts in BM were 6-12 lm in diameter, and the nuclear-cytoplasmic ratio ranged between 0.5 and 0.9. Cells had round, indented, or irregular nuclei with slightly condensed chromatin and small to medium-sized nucleoli. In the cytoplasm, variable amounts of mitochondria, rough endoplasmic reticulum (ER), and Golgi apparatus were found among the cells. Twenty to 30% of leukemia blasts contained granules that were 0.3-1.2 mm in diameter with a variable appearance, representing immature basophils ( Fig. 3A and B) [8][9][10]. The materials in the granules showed a similar electron density to the cytoplasm, more intense electron density, or a speckled appearance, and the materials were often extracted to display an electronlucent appearance. Occasional granules contained myelinlike structures. Theta granules were not apparent. On the other hand, approximately 40% of the cells had primary granules of 0.25-0.6 mm in diameter, and MPO activity in these cells was detected in the nuclear cistern, rough ER, Golgi apparatus, and/or primary granules, showing the features of myeloblasts (Fig. 3C). In contrast, the contents of the granules as well as cytoplasmic organelles in immature basophils lacked MPO activity ( Fig. 3C and D). G-banding of the metaphase spreads obtained from the BM specimen revealed the der(21)t(16;21)(p11;q22) chromosome, while the reciprocal der(16)t(16;21)(p11;q22) was deleted. The karyotype according to the ISCN (2013) [11] was 45,XY,À16,der(21)t(16;21)(p11;q22) [12] (Fig. 4A). A reverse transcriptase-mediated polymerase chain reaction (RT-PCR) using the nested primer pairs for the FUS-ERG fusion transcripts generated three species of DNA, with molecular sizes of 255, 211, and 176 bp, as described earlier (Fig. 4B) [12]. The products were subjected to direct sequencing, demonstrating the in-frame junction encompassing FUS exon 7 and ERG exon 10 (Fig. 4C).

Treatment and Outcomes
Although the initial treatment regimen consisting of idarubicin and cytarabine failed to eradicate leukemia cells, MEC (mitoxantrone, etoposide, and cytarabine) salvage treatment led to a hematological response. Cellulitis of the leg resolved during the induction treatment. After three cycles of MEC, FUS-ERG fusion mRNA fell to below the level of detection. The patient then underwent hematopoietic stem cell transplantation from his daughter, who was HLA-A locus mismatched in the graft-versus-host direction and HLA-C and DR loci-mismatched in the host-versus-graft direction. Although the course of the transplant was uneventful, his leukemia briefly relapsed on day 56 of transplantation. A reduction in the dosage and withdrawal of tacrolimus led to the elimination of circulating leukemia blasts; however, he developed severe graft-versus-host disease involving the entire gastrointestinal tract. He finally died of Pseudomonas aeruginosa septicemia on day 419 of the initial presentation.

Discussion
We herein described a patient with a rare type of AML characterized by the marked basophilic differentiation of leukemia blasts and the t(16;21)(p11;q22)/FUS-ERG fusion gene. Basophilic features were clearly demonstrated by (1) cytomorphological differentiation from agranular blasts to mature basophils, (2) toluidine blue metachromasia, (3) the expression of mature basophilassociated antigens, that is CD123 and CD203c, and (4) the presence of characteristic granules under electron microscopy.
Due to the paucity of ABL and AML with t(16;21), no guidelines for the treatment of this particular patient were available. Although leukemia was refractory to the idarubicin and cytarabine induction treatment, MEC salvage treatment led to remission at the molecular level. However, FUS-ERG RT-PCR briefly became positive, and after allogeneic hematopoietic stem cell transplantation, the disease showed florid relapse. Thus, it became apparent that currently available treatments for AML were unable to eradicate leukemia cells. FUS-ERG was very recently shown to act as a transcriptional repressor of the retinoic acid signaling pathway and the treatment of t(16;21)-AML cells with all-trans retinoic acid led to reductions in the viability and differentiation of these cells [28]. Thus, a clearer understanding of the molecular pathways affected by FUS-ERG will facilitate the development of effective target therapy.

Authorship
YT: main author, was responsible for writing and reviewing the manuscript, literature review, and provided direct care to the patient. YN: was responsible for writing and reviewing the manuscript. DS: performed electron microscopic analysis. CK: performed cytogenetic analysis. KT: performed cytomorphological analysis. KF: performed RT-PCR and direct sequencing of FUS-ERG fusion gene. MH: performed flow cytometric analysis. HO: was responsible for writing and reviewing the manuscript, literature review, and overall revision of the manuscript.