Klinefelter syndrome (KS), with an incidence of 1 in 600 male newborns, is the most
common type of X chromosome aneuploidy. Individuals with KS are characterized by tall
stature, decreased secondary sexual characteristics, small testicles, gynecomastia,
and infertility. About 80% of patients have the karyotype 47,XXY [1]. An extra X chromosome
as a sole acquired abnormality has also been reported in patients with several hematologic
malignancies such as acute lymphoblastic leukemia [2], AML [3, 4], and chronic neutrophilic
leukemia [5]. It has been suggested that an extra X chromosome has high oncogenic
potential, predisposing the carriers to leukemia. In practice, it is sometimes difficult
to distinguish whether the extra X chromosome is constitutional or acquired in a patient
with hematologic malignancy. Here, we describe a mosaic KS patient who was diagnosed
with primary myelofibrosis, which progressed to AML. Because of the obscure clinical
phenotype, we originally reported his sex chromosome aneuploidy as an acquired anomaly
secondary to his hematologic malignancy.
A 62-yr-old man presented with pancytopenia with sustained fatigue, poor general condition,
and excessive weight loss (10 kg in 2 months). His complete blood count revealed the
followings: hemoglobin, 7.4 g/dL; leukocyte count, 3.45×109/L (absolute neutrophil
count, 0.93×109/L); and platelets, 42×109/L. In addition, analysis of the peripheral
blood revealed 3% myeloblasts and leukoerythroblastic features. The patient had mild
splenomegaly on the abdominal computed tomography scan. For further evaluation, the
patient underwent bone marrow aspiration and biopsy, and the results suggested primary
myelofibrosis (PMF), fibrotic stage, with diffuse bone marrow fibrosis that was evident
on Masson-trichrome and reticulin staining. The estimated cellularity of the bone
marrow section was 100%. JAK2 V617F mutation analysis was negative. Conventional cytogenetic
evaluation of the peripheral blood lymphocyte culture using conventional G-banding
revealed 2 cell lines, 47,XXY and 46,XY, with the dominant karyotype being 47,XXY
(Fig. 1). Clinical investigation revealed normal-sized testes, masculine pubic and
axillary hair, no gynecomastia, average height (174 cm) and weight (75 kg), and a
normal serum testosterone level (0.75 µIU/mL; cut-off level, 0.35-5.5 µIU/mL). The
patient was married and fathered a child, inconsistent with KS. Therefore, we reported
the karyotype of 47,XY,+X[14]/46,XY[2] as an acquired anomaly rather than a constitutional
abnormality. Two months later, the blasts in the patient's blood increased up to 47%,
and marrow examination revealed a packed marrow with myeloperoxidase (MPO)-positive
blasts and diffuse fibrosis, as observed before. Flow cytometric immunophenotyping
revealed blasts positive for CD33, CD117, CD11c, CD64, CD56, and MPO. These findings
were consistent with a diagnosis of AML with monocytic differentiation. At the time
of AML transformation, the chromosome analysis still showed the 47,XXY karyotype in
all 23 metaphase cells analyzed. After induction chemotherapy, the patient achieved
complete remission; however, the karyotype abnormality did not disappear.
To evaluate the persistent cytogenetic abnormalities, we further investigated lymphocytes
and buccal mucosa cells using sex chromosome-specific probes that targeted the α-satellite
of the X centromere region and satellite III of Yq12 (CEP X, Spectrum Orange; CEP
Y, Spectrum Green) as described by the manufacturer (Abbott Molecular, Abbot Park,
IL, USA). These probes were hybridized to interphase cells and visualized by fluorescence
microscopy using FISH. The number of individual cells was expressed as a percentage
of the total number of interphase cells analyzed. A specimen that contains >2.30%
of cells with a signal pattern other than XY in male patients was considered to have
an abnormal complement of sex chromosomes. The FISH results for 200 interphase nuclei
from buccal smear cells were as follows: 86% XXY, 11.5% XY, and 2.5% XX (Fig. 2).
With the exception of the XX signals, the proportions of XXY and XY signals in lymphocytes
were similar to those observed in buccal cells (91.2% and 8.8%, respectively). The
XX signal pattern observed in the buccal smear cells was probably due to the artificial
loss of 1 Y chromosome during the FISH procedure or true mosaicism with 3 cell lines
[6]. Consequently, the patient was diagnosed with mosaic KS.
The classical phenotype of KS is widely recognized, but some affected individuals
have no discrete clinical features, especially in mosaic KS. They may have normal-sized
testes and less severe endocrine abnormalities; further, they may be fertile because
of the presence of some normal clones of cells within the testes, as observed in our
case [7]. The variation in phenotype most likely depends on the number of abnormal
cells and their location in the body [8]. Consequently, the disorder might be underdiagnosed;
only approximately 25% of adult men with KS are diagnosed [9], and the referring primary
or secondary centers do not suspect 60% of KS patients to have the disorder, despite
previous clinical investigations [10].
There are several reports of hematologic malignancies with unusual, sole X chromosome
aberrations [2-5, 11-13]. These observations suggest that we should be careful when
concluding whether an abnormality is constitutional or acquired, especially when the
patient has a hematologic malignancy. To prevent misinterpretation, it can be helpful
to perform serial cytogenetic evaluation. If an initial abnormal karyotype returns
to a normal female or male karyotype after chemotherapy, then the initial cytogenetic
abnormality represents an acquired aberration rather than a constitutional one. Careful
clinical investigation should be performed in every case, although general features
should be regarded with caution because patients with chromosomal mosaicism commonly
show very few clinical symptoms, as described in this case. Another approach can be
the use of complementary diagnostic methods, such as interphase FISH. Although cytogenetic
analysis of peripheral blood lymphocytes is the gold standard to confirm KS, interphase
FISH for different somatic cell lines (for example, buccal cells, skin fibroblasts,
or testicular biopsy samples) can be used to confirm chromosomal mosaicism when cytogenetic
analysis of peripheral blood reveals a normal male karyotype in a patient with suspected
KS [7]. FISH is more accurate in determining the exact number of sex chromosomes,
defining the cytogenetic status as mosaic or nonmosaic, and assessing the ratios of
cell populations in mosaicism [14]. In conclusion, we described a mosaic KS patient
who had PMF with AML transformation. Chromosomal mosaicism was confirmed by additional
FISH analysis of the buccal smear cells. We suggest that interphase FISH should be
performed in different somatic cells in order to determine the cytogenetic status
of a patient with suspected KS, especially when an extra X chromosome is the only
abnormality.