ABSTRACT
Erythroid sarcoma is very rare form of pure erythroid leukemia with
undetermined biological features. Here, we present an infant with a
multifocal erythroid sarcoma, diagnosed because the tumor cells were
positive for glycophorin A. After acute myeloid leukemia-oriented
chemotherapy and surgical resection followed by cord blood
transplantation, he has successfully maintained complete remission
without any late effects. Total transcriptome analysis of the tumor
identified a novel fusion gene, RCC1-LCK , and high LCKexpression levels, suggesting that LCK overexpression was involved in
leukemogenesis in this case.
INTRODUCTION
Pure erythroid leukemia (PEL) presenting as erythroid sarcoma (ES) is
extremely rare in children.1-6 Due to the rarity of
PEL and ES in childhood, genetic studies of such cases are scarce.
In this report, we identified a novel kinase-related fusion gene,RCC1-LCK , in an infant case with PEL presenting as ES, which
provides insight into the leukemogenic mechanism in this case.
CASE REPORT
A 6-month-old infant was referred to our hospital because humeral
osteolysis was unexpectedly detected when he was examined for fever.
Laboratory examination of peripheral blood showed the following:
hemoglobin, 9.2 g/dL; platelet count, 310,000/μL; leucocyte count,
11,600/μL, without blasts; C-reactive protein, 15.82 mg/dL; lactate
dehydrogenase, 892 U/L; and ferritin, 922 ng/mL. Computed tomography
showed multiple tumors in the left humerus, intracranium, orbita, and
chest, and beside his spinal cord. Bone marrow examination revealed
infiltration of tumor cells with round coarse nuclei and plentiful
basophilic cytoplasm, sometimes with cytoplasmic budding. Tumor cells
also frequently contained several cytoplasmic vacuoles, but not
Auer-bodies or cytoplasmic granules. Tumor cells were negative for
myeloperoxidase, chloroacetate esterase, and Periodic acid-Schiff (PAS)
on cytochemical staining. Flow cytometry (FCM) analysis showed that they
expressed only CD36, without CD1a, CD14, CD45, CD56, HLA-DR, or
glycophorin A (GPA), resulting in no definite diagnosis.
Immunohistochemistry analysis of a bone marrow clot revealed negativity
for CD3, TdT, CD20, CD138, synaptophysin, chromogranin A, CD99, CD30,
and ALK. Karyotype analysis detected 46, XY in 20/20 metaphases and
fluorescent in situ hybridization did not detect split signals forALK , KMT2A , or EWSR1 . Since acute symptomatic
seizures caused by intracranial tumors were observed, we were obliged to
administer a single dose of vincristine, resulting in rapid shrinkage of
the intracranial tumors detected by brain magnetic resonance imaging
(MRI). A month later, he had symptoms of acute flaccid paralysis of both
lower extremities. Spine MRI revealed recurrence of a paraspinal tumor
that compressed the spinal cord at Th9-L1. Cerebrospinal fluid
examination revealed infiltration of tumor cells, while no tumor cell
infiltration was detected on bone marrow aspiration. FCM analysis of
resected paraspinal tumor cells was positive for CD36 and GPA. Culture
of excised tumor cells in liquid medium for several days led to marked
formation of intracellular vacuoles (Fig. 1A), and some tumor cells
showed granular to diffuse positive results on PAS staining (Fig. 1B).
FCM analysis revealed that the cultured cells were positive for CD71
(transferrin receptor), CD99, CD36, and GPA (Fig. 1C), as well as blood
group A antigen, consistent with the patient’s blood type (Fig. 1C).
Based on these findings, a diagnosis of PEL presenting as ES was finally
made.
He received multi-agent chemotherapy, consisting of cytarabine,
anthracyclines, and etoposide, including intrathecal injection of
cytarabine, methotrexate, and hydrocortisone.7 After
induction therapy, the residual tumors shrank somewhat; however, ES
relapsed with an epidural tumor of the spinal cord at L1-2 after the
second consolidation therapy. No bone marrow relapse was detected. He
underwent tumor resection, followed by allogeneic cord blood
transplantation (CBT), with 5/6 HLA matching at the antigen level. The
conditioning regimen consisted of fludarabine, busulfan, and melphalan.
He has maintained complete remission for 20 months after CBT and
currently has no late effects.
To investigate the underlying pathogenesis of this unique case, we
performed whole-exome sequencing and total transcriptome analysis of the
tumor. Although whole-exome sequencing did not detect any pathogenic
genetic mutations, total transcriptome analysis identified a novel
fusion gene, RCC1-LCK . Fusion of the 5’ end of RCC1(NM_001048199.3) exon 2 and the 3’ end of LCK (NM_001330468.2)
exon 2 was confirmed by reverse transcriptase PCR and Sanger sequencing
(Figs. 2A and 2B). In this fusion gene, the 5’ untranslated region ofRCC1 was joined to the entire coding region of LCK ,
resulting in swapping of the LCK promoter, due to
intrachromosomal deletion (Fig. 2C). Consistent with this promoter
swapping, transcriptome analysis revealed a fragments per kilobase of
exon per million mapped reads (FPKM) value for LCK of 23.2, which
was much higher than that in hematological malignancies withoutLCK rearrangement (FPKM values ranging from approximately 0 to
1).
DISCUSSION
A previous comprehensive genomic analysis of pediatric acute erythroid
leukemia (AEL) showed alterations of several genes; however, the
majority were not common to more than one case, suggesting genetic
heterogeneity in this leukemia subtype and no LCK alterations
were reported in AEL, including PEL.8 Only six cases
of pediatric PEL have ever been reported to have rearrangements, which
include NFIA-CBFA2T3 , NFIA-RUNX1T1 , andZMYND8-RELA ;5,6,9-12 however, the precise
leukemogenic mechanisms underlying the fusion genes in these cases
remain unknown.
Here, we present a case report of a very rare pediatric patient with ES
carrying an RCC1-LCK fusion gene. Given the high FPKM value ofLCK , overexpression of LCK may have contributed to leukemogenesis
in this case. The molecular mechanism underlying overexpression of LCK
is promoter swapping; as a result of the translocation, the
constitutively active promoter of the partner gene drives ectopic
expression of LCK.13
Regulator of chromosome condensation 1 (RCC1) is a known guanine
nucleotide exchange factor of RAN, a nuclear RAS-like G protein, which
is ubiquitously expressed in more than 25 organs and associated with the
cell cycle, DNA damage, and oncogenesis. High expression of RCC1 acts as
a pathogenic partner, promoting the development of some tumors, such as
lung adenocarcinoma and cervical cancer.14
Lymphocyte-specific protein tyrosine kinase (LCK) is a member of the SRC
family of protein tyrosine kinases and a key molecule in regulation of
T-cell functions; for example, LCK regulates the initiation of TCR
signaling, T-cell development, and T-cell
homeostasis.15 Further, LCK expression is reported to
be high in hematological malignancies, such as chronic lymphocytic
leukemia, B-cell acute lymphoblastic leukemia (ALL), and acute myeloid
leukemia (AML), and even in non-hematological malignancies, such as
breast, colorectal, and lung cancers.16 In addition,
LCK functions as both an important signaling molecule and a therapeutic
target in ALL and AML.17-20 Duque-Afonso et al. showed
that suppression of LCK expression decreased the phosphorylation of
PLCγ2 in a form of B-ALL with LCK overexpression, and that PLCγ2 had a
pathogenic role in this form of leukemia, while inhibition of LCK
reduced leukemic cell growth in vitro and in vivo.17In T-ALL, Gocho et al. reported that tumors with high LCK activity were
more sensitive to dasatinib than those with low LCK
activity.18 Further, in AML, Rouer et al. showed that
high expression of LCK was present in leukemic cells from patients with
less differentiated AML, and that a normal LCK expression pattern was
restored when complete remission was achieved.19Intriguingly, similar to T-ALL, Li et al. reported that
LCK-overexpressing AML cells (CTV1) were strongly inhibited by several
LCK inhibitors, including dasatinib.20 Hence, although
the dysregulation of LCK in PEL or ES has not been reported, LCK
overexpression via promoter swapping is potentially a key factor in our
case, and dasatinib may be an effective therapy option for such cases.
In conclusion, we present a diagnostically challenging and unique case
of an infant with ES harboring a novel RCC1-LCK fusion gene. As
the precise leukemogenic mechanism underlying LCK upregulation in PEL or
ES is unknown, further studies are required to clarify the role of LCK
overexpression in this case.