Download Gain of multiple copies of the CBFB gene: a new genetic

Cancer Genetics and Cytogenetics 179 (2007) 62e65
Short communication
Gain of multiple copies of the CBFB gene: a new genetic
aberration in a case of granulocytic sarcoma
Mar Malloa,b,*, Blanca Espineta, Marta Salidoa, Ana Ferrera, Carmen Pedroc,
Carles Bessesc, Encarna Pérez-Vilaa, Sergi Serranoa,
Lourdes Florensaa, Francesc Soléa,b
a
Laboratory of Cytogenetics and Molecular Biology and Laboratory of Hematological Cytology (URNHE-IMAS/IMIM
and URTTS-IMAS/IMIM), Pathology Service, Hospital del Mar, Passeig Marı́tim, 25-29, 08003 Barcelona, Spain
b
Department of Cell Biology, Physiology, and Immunology, Faculty of Life Sciences, Autonomous University of Barcelona,
Bellaterra, Barcelona, Spain
c
Clinical Hematology Service, Hospital del Mar, Barcelona, Spain
Received 8 May 2007; received in revised form 27 July 2007; accepted 30 July 2007
Abstract
Granulocytic sarcomas (GS) are tumor masses of immature myeloid cells presenting at an extramedullary site, mainly the skin, bone, and lymph node. They are often associated with acute myeloid
leukemia (AML) with monoblastic or myelomonocytic differentiation, including either AML M2
with t(8;21)(q22;q22) or AML M4Eo with inv(16)(p13q22). We present a case diagnosed with
GS associated with AML M4 that presented a normal karyotype with conventional cytogenetic
analysis. Although the myeloblasts did not show the inv(16)(p13q22) (CBFB/MYH11), a gain of
multiple copies of the CBFB gene was detected with fluorescence in situ hybridization analysis.
To our knowledge, no cases with this rare genetic anomaly have been previously described. Ó 2007 Elsevier Inc. All rights reserved.
1. Introduction
Myeloid sarcomas, extramedullary myeloid tumors, or
granulocytic sarcomas (GS) are tumor masses of immature
myeloid cells in an extramedullary site. These tumors may
develop de novo or concurrently with acute myeloid leukemia (AML), myeloproliferative disorders, or myelodysplastic syndromes. GS may be the first evidence of AML, or
may precede it by months or years. It can also represent
the initial manifestation of relapse in a previously treated
AML in remission [1,2].
GS are often asymptomatic, depending on their location,
and may therefore remain undiagnosed [3]. The most common sites of occurrence are subperiosteal bone structures,
lymph nodes, and skin [1]. When GS affects the female
genital tract, the organ most commonly involved is the
ovary, followed by the cervix and the uterus [4,5].
Regarding AML subtypes, GS are usually associated
with monoblastic and myelomonocytic acute myeloid
* Corresponding author. Tel.: þ34-93-2483521; fax: þ34-93-2483131.
E-mail address: [email protected] (M. Mallo).
0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.cancergencyto.2007.07.018
leukemias. GS has been described in association with a variety of chromosomal abnormalities [6,7]. In particular,
translocation t(8;21)(q22;q22) is regarded as a recurrent abnormality in most cases identified as AML M2 according to
the FrencheAmericaneBritish (FAB) classification [1,8].
In contrast, only a few cases of inv(16) AML with GS have
been reported [9]. This type of leukemia, usually FAB type
AML M4Eo, is associated with either inv(16)(p13q22) or
t(16;16)(p13;q22), the resultant chimeric gene being
CBFB/MYH11 [10]. CBFB, located at 16q22, is the beta
subunit of the core binding factor, which is a transcription
factor that regulates the expression of essential genes for
normal hematopoiesis [11].
Pileri et al. [2] analyzed a series of 92 cases of GS. Using fluorescence in situ hybridization (FISH) techniques,
they observed clonal abnormalities in 25 out of 49 GS
analyzed. Two different aberrations in two cases were
observed: 20q associated with CBFB deletion and trisomy
4 in conjunction with 5q. Moreover, they presented a case
of GS with uterine and breast involvement, preceding AML
M4, that was characterized by trisomy 8 (revealed by FISH),
and another one with a rare t(1;9)(q11;q34) in the absence of
M. Mallo et al. / Cancer Genetics and Cytogenetics 179 (2007) 62e65
þ8 (revealed by cytogenetic analysis of peripheral blood)
[2].
Here we present the case of a patient with the diagnosis
of GS, which exhibited a gain of multiple copies of the
CBFB gene detected by FISH.
2. Case report
A 50-year-old woman was seen in the gynecology department of Hospital Verge Del Toro, Menorca, Spain, in
July 2005 referring metrorrhagia, malaise, bone pain, and
subcutaneous tumors in abdomen, limbs, and breasts.
Neither lymphadenopathies nor visceromegalies were observed. A uterine mass was detected; biopsy revealed the
presence of immature myeloid cells. The diagnosis of GS
of the uterus was established.
The patient was referred to the hematology department
of our hospital. The main laboratory findings (with normal
values) were as follows: hemoglobin 111 g/L (130e170
g/dL), hematocrit 34% (40e54%), mean corpuscular volume 105 fL (82e97 fL), platelet count 167 109/L
(150e450 109/L), and white blood cell count 4.3 109/L (4e11 109/L) without blast cells and lactate dehydrogenase serum level 760 IU/L (!450 IU/L). The hemostatic balance was normal.
The bone marrow aspiration was hypercellular and
showed predominance of two populations: neutrophilic
(35%, with 10% of blast cells showing Auer rods) and
monocytic (31%, with 15% of blasts monocytoid). Cytochemistry study showed a result compatible with myeloid
blasts. No eosinophilia was found.
Flow cytometry analysis of blast cells disclosed the presence of two populations. The first was positive for CD45
(weak), CD34, CD117, CD7, CD33, and CD15. The second
Fig. 1. Fluorescence in situ hybridization (FISH) interphase nucleus
showing three copies (orange/green signals) of the CBFB gene (LSI CBFB
dual-color, break-apart rearrangement probe; Abbott Molecular/Vysis, Des
Plaines, IL) in a case diagnosed as acute myeloid leukemia M4 and
granulocytic sarcoma (orange/green signals).
63
was strongly positive for CD45, CD33, and CD15 and also
expressed CD4, CD11b, and CD14; in addition, it was
negative for CD34, CD7, and CD17. Both populations
showed myeloperoxidase positivity. These results were in
accordance with the presence of immature cells with myelomonocytic differentiation. The diagnosis of acute myelomonocytic leukemia (AML M4) subtype according to the
FAB classification was established.
Conventional banding cytogenetic study was performed
at diagnosis on a 24-hour bone marrow cell culture. Twenty
metaphases were analyzed, and all showed a normal
karyotype.
To rule out the pericentric inversion of chromosome 16,
characteristic of AML M4 with eosinophilia (AML M4Eo),
we performed FISH with LSI CBFB dual-color, break-apart
rearrangement probe (Abbott MoleculareVysis, Des
Plaines, IL). We did not find cells displaying the inv(16),
but did observe that, in 200 nuclei analyzed, 75% presented
multiple copies of the CBFB gene: 49% with three copies,
15% with four copies, and 11% with five copies. A representative example of the CBFB gene gain of multiple copies
is shown in Figure 1.
To confirm that the CBFB gene gain of multiple copies
observed was not a gain of the whole chromosome 16,
we applied FISH with an IGH/MAF dual-color, dual-fusion
probe (Abbott Molecular/Vysis). This probe labels the MAF
gene (16q23) in SpectrumOrange and the IGH gene
(14q32) in SpectrumGreen. We observed only two copies
of MAF (Fig. 2), suggesting gain of multiple copies of just
the CBFB gene.
Reverse transcriptaseepolymerase chain reaction (RTPCR) analysis was performed to detect either CBFB/
MYH11 or RUNX1 (previously AML1)/RUNX1T1 (alias
ETO) rearrangements, and was negative for both transcripts. Moreover, an analysis of the FLT3 gene duplication
excluded the presence of duplication of the JM domain.
The patient received chemotherapy with IDICE (idarubicin, cytarabine, etoposide, and CSF) regimen and achieved
a complete remission. At that time, new cytogenetic and
FISH studies were performed. Conventional banding cytogenetic revealed a normal karyotype, and FISH analysis
showed a gain of multiple copies of the CBFB gene, with
a lower number (1.8% with O3 copies) of cells showing
more copies of CBFB than those observed at diagnosis.
These results are within normal for the FISH technique.
A consolidation treatment with mitoxantrone and cytarabine was administered.
After this treatment, a new FISH analysis revealed persistence of the CBFB gene gain of multiple copies. Table 1
shows the results of conventional banding cytogenetic and
FISH studies performed during the course of the disease.
Once the patient had finished the chemotherapeutic
treatment, she underwent an allogeneic hematopoietic stem
cell transplantation with a reduced-intensity conditioning
regimen. However, she relapsed and died 7 months after
diagnosis.
M. Mallo et al. / Cancer Genetics and Cytogenetics 179 (2007) 62e65
64
Fig. 2. FISH interphase nucleus with two green signals (IGH gene) and two orange signals (MAF gene), indicating no gain of the whole chromosome 16.
3. Discussion
GS has been described in association with a variety of
chromosomal abnormalities [6,7]. GS occurs in ~18% of
patients with t(8;21) AML [12]. Nevertheless, to our
knowledge only 12 cases of inv(16) have been reported
since 1988. In 11 of the cases, GS was located in the abdominal cavity (small intestine, ileum, mesentery, ovary,
rectum, and jejunum); none of the 12 cases involved the
uterus. Although there are several differences between lesions observed in t(8;21) and inv(16) AMLs, the pathogenesis could be related to regulation of CBFB transcription
factor, involved in cell recognition and adhesion, thus
resulting in leukemia infiltration [9]. Pathak et al. [13]
reviewed 25 cases of GS of the cervix; three of them
represented a relapse of an AML and nine of them were,
after review, diagnosed as AML [13]. Recently, Garcia
et al. [14] described 11 cases of GS involving the gynecologic tract; in 8 of them, the uterus was affected.
Here, we have described the case of a patient diagnosed
as having a GS of the uterus and, simultaneously, with
an AML M4. She did not exhibit the pericentric inversion
Table 1
Conventional cytogenetics karyotype in a woman diagnosed with
granulocytic sarcoma, with fluorescence in situ hybridization analysis
of interphase nuclei for the CBFB gene
Nuclei with copies of CBFB, %
Date (2005) Clinical status Karyotype 3 copies
4 copies
5 copies
October
November
December
15
0.2
1.5
11
0
0
Diagnosis
CR
CR
46,XX[20] 49
46,XX[20] 1.6
ND
0
All samples were bone marrow.
Abbreviations: CR, complete remission; ND, not done.
of chromosome 16 [inv(16)(p13q22)], but FISH analysis
revealed a gain of multiple copies of the CBFB gene.
It is often difficult to determine the chromosomal rearrangement of inv(16) by conventional banding cytogenetics, due to the presence of metaphase preparations of
suboptimal quality in AML [15]. In this regard, molecular
analysis with RT-PCR and FISH techniques are useful in
allowing a precise diagnosis [16,17]. In the present case,
we would not have detected the CBFB gain of multiple copies without the FISH analysis. This aberration might be
present in other similar cases, but could go unnoticed unless
FISH techniques are applied.
A normal karyotype in AML is suggestive of good prognosis. The reported case had no alterations revealed with
conventional banding cytogenetics, but FISH analysis did
reveal an aberration: a CBFB gene gain of multiple copies.
This finding could help explain the prognosis and the clinical evolution of the patient. Nonetheless, any further cases
with this rare cytogenetic aberration should be reported, in
order to add to the understanding of the diagnosis and clinical implications.
For identifying the CBFB gene gain of multiple copies, we
were not able to analyze a biopsy of the uterus tissue. Initially,
the patient was diagnosed with GS and later with AML M4.
The FISH study was performed on the bone marrow sample.
Based on previous reports, we assume that AML and GS are
the same lesion and so, in consequence, the genetics are the
same for both. The literature does offer some confirmation
of this idea. Lillington et al. [18] presented a cytogenetic
and molecular study that showed evidence of marrow involvement in a GS. They studied tissue from a subcutaneous
nodule and bone marrow and they identified the same alteration: an in-frame fusion of the MLL and MLLT10 (alias
M. Mallo et al. / Cancer Genetics and Cytogenetics 179 (2007) 62e65
AF10) genes (interchromosomal exchanges between chromosomes 10 and 11). In this case, they also observed an intrachromosomal rearrangement of chromosome 5 [18].
Lee et al. [19] described a case of GS presenting as
pneumonia in a child with t(8;21) AML (AML M2). They
studied a lung biopsy specimen with FISH and identified
a t(8;21)(q22;q22), which confirmed the retrospective diagnosis of a well-differentiated pulmonary GS [19].
Finally, Al-Quran et al. [20] presented a case of GS
of the urinary bladder and epididymis as a primary manifestation of AML with inv(16). They performed conventional cytogenetic studies of marrow and demonstrated
inv(16)(p13q22) in 4 of 20 metaphases. In addition, they
studied the inv(16) by FISH in the bladder neoplasm. They
confirmed the evidence of an associated myeloid neoplasm
in marrow [20].
To better characterize the role of CBFB gene gain of
multiple copies, we strongly recommend applying FISH
techniques for patients with GS.
To our knowledge, we have presented here the first reported case of CBFB gene amplification in a patient diagnosed with AML M4 presenting with GS in the uterus, an
aberration that has not been previously described, neither
in GS nor in other pathologies.
Acknowledgments
This work was supported in part by grants from ‘‘Instituto de Salud Carlos III’’ of Spain (C03/07 and C03/10).
We thank Carme Melero for her excellent technical
assistance.
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