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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2886-2892
Fluorescence In Situ Hybridization of Progenitor Cells Obtained
by Fluorescence-Activated Cell Sorting for the Detection of Cells
Affected by Chromosome Abnormality Trisomy 8 in Patients With
Myelodysplastic Syndromes
By
Kohki Saitoh,
Ikuo Miura,
Naoto Takahashi, and
Akira B. Miura
From the Third Department of Internal Medicine, Akita University
School of Medicine, Akita, Japan.
 |
ABSTRACT |
Myelodysplastic syndrome (MDS) is believed to be a stem-cell
disorder involving cytopenia and dysplastic changes in three hematopoietic lineages. However, the involvement of pluripotent stem
cells and progenitor cells has not been clarified conclusively. To
address this issue, we used fluorescence in situ hybridization (FISH)
of blood and bone marrow (BM) smears for mature cells and FISH of cells
sorted by fluorescence-activated cell sorting for progenitor cells.
Seven patients with MDS associated with trisomy 8 were studied. FISH
showed +8 in granulocytes, monocytes, and erythroblasts, but not in
lymphocytes. Sorted cells of T (CD3+), B
(CD19+), and NK cells
(CD3 CD56+) from peripheral blood did not
contain +8, nor did CD34+ subpopulations from BM
including B (CD34+CD19+), T/NK
(CD34+CD7+) progenitors, and pluripotent
stem cells (CD34+Thy1+). The +8
chromosome abnormality was identified in stem cells only at the level
of colony-forming unit of
granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM;
CD34+CD33+). It may thus be concluded that
cells affected by trisomy 8 in the context of MDS are at the CFU-GEMM
level and that cells of lymphoid lineage are not involved. These
results provide new insights into the biology of MDS and suggest that
intensive chemotherapy and autologous BM transplantation may become
important therapeutic strategies.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
MYELODYSPLASTIC SYNDROME (MDS) represents
a group of acquired hematopoietic disorders. In 1982, the
French-American-British (FAB) Cooperative Group proposed a
morphological classification of these disorders into five categories:
refractory anemia (RA), RA with ring sideroblasts (RARS), RA with an
excess of blasts (RAEB), chronic myelomonocytic leukemia (CMML), and
RAEB in transformation (RAEB-T).1 MDS is characterized by
cytopenia of more than two hematopoietic lineages with normal or
hyperplastic marrow and dysplastic changes in three lineages. Because
of the dysplastic changes, MDS has been thought to be a stem-cell
disorder. Previous studies with glucose-6-phosphate dehydrogenase
(G-6-PD),2 chromosome analysis,3
restriction-length polymorphism (RFLP) analysis of phosphoglycerate
kinase (PGK), and hypoxanthine phosphoribosyl transferase
(HPRT)4 support the hypothesis that MDS is a clonal disorder at a multipotent hematopoietic stem cell level. Our study yielded data consistent with involvement of the myeloid series but not
of the lymphoid lineage.
From 60% to 73% of patients with MDS have nonrandom chromosome
abnormalities. Trisomy 8, monosomy 7/7q-, monosomy 5/5q-, and 20q- are
frequently found.5-7 The feasibility of chromosome analysis of every cell lineage is limited when standard cytogenetic techniques are used. A major disadvantage of G-6-PD isoenzyme analysis is the
rarity of heterozygosity outside certain ethnic groups. Polymerase chain reaction (PCR) studies require highly purified cell suspensions and are hampered by cell contaminants. In contrast, fluorescence in
situ hybridization (FISH) enabled us to identify chromosomal changes in
nondividing cells of every lineage and to perform a cell by cell
analysis in addition to analysis of metaphase spreads.8-13 Furthermore, even a small-cell population can be collected by fluorescence-activated cell sorting (FACS) based on immunophenotype. The major disadvantage of flow cytometry is the lack of direct correlation between morphology and cell markers. For this reason, we
used FISH of blood and bone marrow (BM) smears for mature cells and
FISH of cells sorted by FACS for identification of stem cells in MDS
patients with the typical chromosomal change of trisomy 8. Because this
technique does not require cell culture or amplification, "sorter
FISH" (FACS + FISH) can provide direct analysis of immature progenitor cells.
| |
MATERIALS AND METHODS |
Patients.
Seven patients with MDS and carrying trisomy 8 chromosomes were
selected for this study (Table 1). The
diagnosis of MDS and determination of the subgroups were performed
according to the FAB classification. The patients comprised three men
and four women, whose age ranged from 35 to 73 years. MDS subgrouping
yielded three patients with RA, one patient with RAEB, one patient with CMML, one patient with RAEB-T, and one patient whose disease
changed from RAEB to acute myelocytic leukemia (M2). Patient 5 had a
history of treatment for essential thrombocytosis (ET), and patient 7 had a history of treatment with melphalan and prednisolone for multiple
myeloma from 1987 to 1991; in 1991, pancytopenia and chromosome
abnormality der(1;7) were found and the diagnosis of treatment-related
MDS (t-MDS) was made.
Peripheral blood (PB) specimens and BM were obtained from all patients
after informed consent. BM mononuclear cells (BMMC) or PB mononuclear
cells (PBMC) were isolated by Ficoll-Hypaque density-gradient
centrifugation. After removal of the phagocytic cells from BMMC, the
remaining nonphagocytic cells were used for the following study with
FACS.
Chromosome analysis.
We used a standard technique for chromosome analysis. Aspirated BM (2 × 107 cells/mL) was cultured overnight, and the cells
were exposed to Colcemid (GIBCO-BRL, Gaithersburg, MD)
with 0.02µg/mL 2-hours before harvest. After 15 minutes of hypotonic
treatment with 0.075 mol/L KCl, the cells were fixed with ethanol and
acetic acid (3:1), and G-banded karyotype was analyzed and described
according to the International System for Human Cytogenetic
Nomenclature (ISCN 1995).14
FACS.
Fluorescein isothiocyanate (FITC)-conjugated CD7 (3A1-FITC, Coulter
Immunology, Hialeah, FL), CD19 (B4-FITC; Coulter), CD33 (MY9-FITC;
Coulter), CDw90 (Thy-1-FITC; Coulter), FITC CD3 (Leu4-FITC; Becton
Dickinson, San Jose, CA), and phycoerythrin (PE)-conjugated CD34
(HPCA2-PE), CD56 (Leu19-PE) and CD3 (Leu4-PE; Becton Dickinson, Sunnyvale, CA) were used. Both FITC-conjugated and PE-conjugated nonspecific Ms IgG was obtained from Becton Dickinson.
Flow cytometry analysis and cell sorting were performed on an EPICS
Elite (Coultronics, Merrgency, France). Between 1,000 to 30,000 cells
per fraction were sorted and collected with FACS with a purity of 95%.
These cells were then used to make cytospin preparations and were
stained with May-Grünwald-Giemsa.
FISH.
The probes used in this study were commercially available
chromosome-8-specific probes (D8Z2; Oncor, Inc, Gaithersburg, MD), which were labeled with Digoxigenin. For prehybridization, slides were
immersed in 0.01N HCl/0.005% pepsin for 5 minutes, washed two times in
phosphate-buffered saline (PBS) for 3 minutes, and treated with 4%
paraformaldehyde/PBS for 5 minutes. After two washes with PBS, the
cells were dehydrated through 70%, 80%, and 100% ethanol. The
hybridization protocol followed the manufacturer's instructions.
Signals were visualized by using 0.2 µg/µL
antidigoxygenin-rhodamine (Boehringer Mannheim Biochemica, Mannheim,
Germany)/1% block ace (Dainippon Pharmaceutical, Tokyo, Japan), which
inhibits the nonspecific background. After counterstaining with 0.1 µg/mL 4 , 6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, MO),
the signals were observed under a Nikon microscope (Tokyo, Japan) with
a FITC/rhodamine dual-band filter, G2A, and a UV filter (Nikon). The
preparations were evaluated by counting 100 nuclei per slide. The mean
percentage of nuclei with a false-positive signal was calculated to
arrive at a control PB from hematologically disease-free individuals.
Although a previous study showed false-positive cells occurring at a
frequency of about 2%, ours yielded a frequency of mean + 2SD:4.9%
(cutoff).
Stem cells were characterized by defining their subpopulations of
CD34+ cells. Thy-1 is a marker expressed by fetal and adult
BM stem cells. The Thy-1+ subset has multilineage
differentiation capacity as shown by its ability to produce T cells, B
cells, and myeloid cells.15,16 The
CD34+CD10+CD19+ population
represents exclusively B-lymphoid-committed
progenitors.17 Cytoplasmic CD3+CD7+
cells were considered to represent committed T-cell progenitors based
on the fact that CD2, cyCD3, CD5, and CD7 are coexpressed on all mature
T cells but not on all mature B cells.18 However, these
antigens are also expressed on CD34+ natural-killer (NK)
progenitors.19 In addition, CD7 is also present on B-cell
and myeloid progenitors.20 These data suggest that
CD34+CD7+ and CD5+/CD2+
populations represent T-lymphoid-committed progenitors.18
On the basis of these considerations, we considered
CD34+Thy-1+ cells to be pluripotent stem cells,
CD34+CD19+ to be B-progenitor cells, and
CD34+CD7+ cells to be T/NK-progenitor cells.
| |
RESULTS |
Chromosome analysis.
The patients' characteristics and karyotypes are listed in Table 1.
Phytohemagglutinin (PHA)-stimulated culture of PB showed normal
karyotypes for all patients.
Flow cytometry analysis.
The percentage of CD34+ BM cells increased from 1.5% for
RA and 3.8% for CMML, to 10.6% for RAEB and 10.6% for RAEB-T, and notably 18.3% for transformed MDS (Table
2).
Lineage involvement was identified by FISH analysis of blood and BM
smears.
Cells collected by FACS tend to have reduced cytoplasm, but there is no
difficulty in evaluating signals in the
nuclei. Although the percentage
of cells with the +8 chromosome aberration varied, the percentage in
erythroid, granulocyte, and monocyte lineages was above the cutoff
value. In contrast, lymphocytes did not show trisomy 8 in any of the
patients, whereas erythroblasts tended to have a lower percentage of
cells with the +8 chromosome abnormality (Figs 1 and 2). In particular,
patient 5 had 20% fewer +8 cells than the other patients.
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| Fig 1.
Percentage of cells bearing the +8 chromosomal
aberration in erythroid, granulocytes, monocytes, and lymphocytes based
on FISH analysis applied to blood and BM smears. No BM smear could be
obtained from patient 3. No chromosomal aberrations were found in the
lymphocytes of any patients.
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| Fig 2.
FISH of BM smear of patient 1. (A) An erythroblast, two
neutrophils, and lymphocytes in May-Grünwald-Giemsa stain. (B)
FISH results applied to the smear. Three signals were recognized in the
erythroblast and neutrophils but none in the lymphocytes.
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FISH analysis applied to sorted cells of B, T, and NK cells.
In the lymphoid cells of all patients, that is, B cells
(CD19+), T cells (CD3+), and NK cells
(CD356+), the percentage of cells with
trisomy was below the cutoff value (Table 3
and Fig 3).
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| Fig 3.
Percentage of cells with the +8 chromosomal aberration
in T (CD3+), B (CD19+), and NK
(CD356+) cells in the blood. In all
patients, the lymphocytes were intact with respect to chromosome 8.
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FISH analysis of stem cells consisting of CD34+
subpopulations.
No CD34+ cells could be collected from Patients 2 and 3 (Table 3 and Figs 4 and
5).
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| Fig 4.
FISH of sorted cells based on immunophenotype. (A), (C),
and (E) show T cells (CD3+), B cells
(CD19+), and NK cells
(CD356+), respectively, and (B), (D), (F),
the FISH results for these cell populations. Two signals were observed
in all these cell types.
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| Fig 5.
Percentage of cells with the +8 chromosome aberration
in CD34+ subpopulations. The
CD34+7+ (T/NK-progenitor cells),
CD34+19+ (B-progenitor cells), and
CD34+Thy-1+ (pluripotent-stem cells) of all
patients showed an intact chromosome 8. Trisomy 8 was observed at a
high frequency among CD34+33+ (CFU-GEMM)
subpopulation cells.
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Analysis of CD34+ subpopulations.
CD34+Thy1+ v
CD34+Thy1 subpopulation.
We could collect enough CD34+Thy-1+ cells from
only three patients (Fig 6A and
B). The percentage of cells with the +8 chromosome abnormality in pluripotent stem cells
(CD34+Thy1+) was below the cutoff value, in
sharp contrast to that in CD34+Thy1 cells.
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| Fig 6.
Cells collected by FACS and stained with
May-Grünwald-Giemsa and FISH results for CD34+
subpopulations. (A), (B) CD34+Thy-1+
cells; (C),(D) CD34+CD19+ cells; (E),(F)
CD34+7+ cells; (G),(H)
CD34+CD33+ cells. Three signals were
observed only in the CD34+CD33+
subpopulation cells.
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CD34+CD19+ v
CD34+CD19 subpopulation.
In contrast with CD34+CD19 cells, the
percentage of CD34+19+ cells with trisomy 8 was
below the cutoff value in all patients (Fig 6C and D).
CD34+CD7+ v
CD34+CD7 subpopulations.
The percentage of cells with trisomy 8 in the
CD34+CD7+ subpopulation was clearly lower than
that in the CD34+CD7 subpopulation (Fig 6E,
6F).
CD34+CD33+ v
CD34+CD33 subpopulation.
In addition to the two patients from whom no cells could be collected,
not enough cells could be obtained from patient 5. The
incidence of trisomy 8 in CD34+CD33+ cells was
similar to that in the CD34+Thy,
CD34+CD7, and
CD34+CD19 subpopulations.
CD34+CD33 cells also contained the +8
chromosome abnormality, although the percentage was less than that of
the CD34+CD33+ subpopulation (Fig 6G and H).
There was significant heterogeneity of +8 cells among the seven
patients that is reflected to a certain extent in the percentage of +8
detected by FISH. Despite this heterogeneity, pluripotent stem cells
(CD34+Thy1+), B-progenitor cells
(CD34+CD19+), T/NK-progenitor cells
(CD34+CD7+), all showed values below the cutoff
value. CD34+CD33+ cells, compatible with the
colony-forming unit of granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM), had a higher percentage with the +8 chromosome abnormality than did CD34+CD33 cells, although no
sharp differences were detected between the two cell types. The
CD34+CD33 subpopulation included pluripotent
stem cells, cells of the lymphoid lineage, and cells
developmentally intermediate between pluripotent stem cells and
CFU-GEMM. Because the first two subpopulations, pluripotent stem cells
and the lymphoid progenitor cells, did not have the +8 chromosome
abnormality, the cell population affected by trisomy 8 is assumed
to consist of CD34+ cells that are more immature than
CFU-GEMM (Fig 7).
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| Fig 7.
Lineage involvement of abnormal clone of MDS. Trisomy 8 was not detected in cells of the lymphoid lineage or in
CD34+Thy-1+ cells. The cells affected by
the chromosomal aberration +8 are these assumed to be at the level of
CFU-GEMM.
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| |
DISCUSSION |
MDS is believed to be a stem-cell disorder originating in dysplastic
changes in multiple hematopoietic lineages. MDS only rarely develops
into acute lymphoid leukemia, in sharp contrast to chronic myeloid
leukemia (CML), a typical stem-cell disorder. CML develops into an
acute phase involving myeloid, lymphoid, and other lineages. To
investigate stem-cell involvement in MDS, we applied FISH to blood and
BM smears for analysis of mature cells and to sorted cells for
progenitors cells.
Cells affected by trisomy 8 in MDS were detected at the level of
CFU-GEMM or an earlier cell population, but not in pluripotent stem
cells. The lymphoid progenitor cells sorted by FACS did not show the +8
chromosome abnormality either. Data from standard chromosome analysis
of PHA-stimulated cultures of PB also support these results.
Furthermore, previous reports on other chromosomal abnormalities than
+8 observed in MDS are consistent with our data.8-13 These
data can explain why MDS patients rarely develop acute lymphoblastic
leukemia, and also provide new insights into the biology of MDS and a
rationale for standard remission-induction chemotherapy for MDS
patients before the onset of disease progression. Autologous BM
transplantation may represent a rational approach to treating MDS
patients early after diagnosis if allogeneic BM transplantation can not
be performed because HLA-matched donors are not available.
We conclude that the combination of FACS and FISH ("sorter FISH")
is an effective technique for identification of the lineage involvement
of stem-cell disorders.
| |
FOOTNOTES |
Submitted March 5, 1998;
accepted June 11, 1998.
Address reprint request to Ikuo Miura, MD, Third Department of Internal
Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita 010, Japan; e-mail: ikuo{at}med.akita-u.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract