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Clonal Analysis of Myelodysplastic Syndrome: Monosomy 7 Is Expressed in the
Myeloid Lineage, But Not in the Lymphoid Lineage as Detected by Fluorescent
In Situ Hybridization
By Winald R. Gerritsen, John Donohue, Jan Bauman, Suresh C. Jhanwar, Nancy A. Kernan, Hugo Castro-Malaspina,
Richard J. O’Reilly, and Jean-Henri Bourhis
Conflicting results have been published on whether or not
myelodysplastic syndromes (MDS) affect all cell lineages.
Involvement of myeloid and erythroid cell lineages has been
regularly observed, but it remains controversial whether the
different lymphoid cell lineages are involved. In this study of
eight patients with MDS associated with monosomy 7,
fluorescent in situ hybridization (FISH) was used t o enumerate the chromosomes 7 in interphase cells. With the probe
D721, the rate of false-positive detection of monosomy 7 was
3% 2 2% in normal cells. T- and 6-cell lines were established
from eight patients with MDS and monosomy 7. As determined by FISH in interphase cells, 1.9% (0% t o 3%) of the
cells in the B-cell lines showed one fluorescent spot and 1.1%
(OYot o 2.9%) of the cells in the T-cell lines. These values do
not differ from normal values. However, the possibility that
normal cells were selected when the T- and B-cell lines were
established could not be excluded. Therefore, peripheral
blood cells were obtained, separated according t o surface
markers specific for lymphoid and myeloid cell lineage with a
cell sorter, and analyzed for the expression of monosomy 7
by FISH. Antibodies recognizing T cells (CD3). B cells (CD20),
natural killer (NK) cells (CD57), monocytes and granulocytes
(low and high expression of C D l l b antigen), and myeloid
progenitors (CD33) were used t o separate cells. The expression of monosomy 7 in the T cells, NK cells, and B cells did not
differ from control values. These results in the lymphoid
subpopulations are in stark contrast with the observations in
the myeloid populations; the percentage of cells with monosomy 7 ranged from 9% t o 78% (controls: 6% & 2%) in cells
with low C D l l b expression, 20% t o 89% in cells with a high
expression of the C D l l b antigen (controls: 7% 2 3%). and
23% t o 91% in the CD33 positive cells (controls: 5% f 3%).
The results of this study suggest that monosomy 7 does not
usually affect lymphoid subpopulations but is restricted t o
committed progenitor cells with the capacity t o differentiate
into mature myeloid cells.
Q 7992 by The American Society of Hematology.
M
with a partial deletion of chromosome 13, as reported by
Lawrence et a],’ abnormal metaphases were scored in both
bone marrow samples and EBV-transformed B-cell lines,
while phytohemagglutinin (PHA)-stimulated peripheral
blood lymphocytes showed a normal karyotype. Carbonell
et a16have studied lineages affected by the 5q- syndrome in
four patients. Cytogenetic analysis of marrow cells showed
that three of the four patients had cells with normal
karyotypes, cells with trisomy 8, and cells with an interstitial
deletion of chromosome 5. Cells from lymphocyte cultures
exhibited normal karyotypes in all four cases. Erythroid and
myeloid colonies had the 5q deletion in all four patients.
However, none of the colonies exhibited trisomy 8.
Results of analyses based on standard cytogenetic techniques may be skewed by the fact that these assays evaluate
YELODYSPLASTIC syndromes (MDS) comprise a
group of hematologic disorders characterized by
ineffective hematopoiesis resulting in progressive cytopenia
affecting erythroid, myeloid, and megakaryocytic lineages.
Eventually, 30% to 40% of the patients with MDS will
develop acute myeloid leukemia, while others die from
complications (hemorrhage, infection) caused by a progressive impairment of hematopoiesis.lJ The need for erythrocyte and platelet transfusions as well as the high susceptibility to infections indicates that multiple cell lineages are
involved in the disease.
Several techniques have been applied to document that
clonal abnormalities are present in different cell lineages.
The first study of glucosed-phosphate dehydrogenase (G-6PD) isoenzyme patterns demonstrated the origin of peripheral blood lymphocytes to be clonal in a patient with
sideroblastic anemia. These lymphocytes displayed the
same isoenzyme as the red blood cells (RBCs), granulocytes, and platelets, suggesting that all these lineages were
affected by the disease? In another patient with sideroblastic anemia, similar results were seen. Additionally, EpsteinBarr virus (EBV)-transformed B cells were all of the same
isotype, suggesting that MDS is a clonal disease derived
from pluripotent stem cells. However, the G-6-PD analysis
in the latter patient was discordant with karyotypic results;
although the marrow cells had specificcytogenetic abnormalities, all B-cell lines showed a normal karyotype:
MDS is a disease associated with multiple chromosomal
abnormalities. Deletion of the long arm of chromosome 5
(5q-), monosomy 7, and trisomy 8 are most frequently
o b ~ e r v e d .The
~ , ~ chromosomal abnormalities can be helpful
for identifymg which cell lineages are affected, but the
results that have been reported have not provided consistent information about which cell lineages are involved in
MDS. In two patients with sideroblastic anemia associated
Blood, Vol80, No 1 (July 1). 1992: pp 217-224
~
~
~
~
~
~
~
~~
~
~
~
From the Bone Marrow Transplantation Service, the Departments
of Pediatrics and Medicine, and Laboratory of Cancer Genetics and
Cytogenetics of the Department of Pathology, Memorial SloanKettering Cancer Center, New York, NY;and the Section of Cytometiy,
Institute for Applied Radiobiology and Immunology TNO, RijswQk,
The Netherlands.
Submitted October 15, 1991; accepted March 13, 1992.
N R G . received a 1989 fellowship from the Aplastic Anemia
Foundation of America and a 1990 fellowship jrom the Fogarty
Intemational Center. Additional support was supplied in part by the
Zelda Weintraub Foundation and the Andrew Gafiey Foundation
(J-H.B.). ‘
Address reprint requests to Richard J. O’Reilly, MD, Bone Marrow
Transplantation Service, Memorial Sloan-Kettering Cancer Center,
1275 YorkAve, New York, NY10021.
The publication costs of this article were defayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
OOO6-4971/92/8001-0011$3.00/0
217
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218
GERRITSEN ET AL
only the small fraction of cells that are actively dividing or
are sensitive to mitogenic stimulation. Two studies that
have used the more sensitive technique of restriction
fragment length polymorphism (RFLP) to examine DNA
polymorphism at X-linked loci have suggested a clonal
origin for both myeloid and lymphoid cells in patients with
MDS.9,10 Recently, involvement of both lymphoid and
myeloid lineages has also been indicated in two patients
with MDS examined for mutations in the ras-oncogenes in
different cell subpopulations?Jl In contrast with these
observations, Kere et all2 reported that five patients with
monosomy 7 had two chromosomes 7 in their lymphocytes,
but only one chromosome 7 in their granulocytes and
monocytes using RFLP analysis of polymorphic sequences
on chromosome 7.
Recently, the technique of fluorescent in situ hybridization (FISH) has been developed. Unlike other techniques,
FISH permits enumeration of all nucleated cells for the
presence or absence of chromosomal deletions irrespective
of cycle status. Because a FISH analysis requires only 500 to
1,000 cells, this technique was considered an excellent tool
to study the lineages affected by MDS associated with
monosomy 7. We have used the technique of FISH to assess
the expression of monosomy 7 in cultured T and B cells as
well as in purified T cells, B cells, NK cells, monocytes,
granulocytes, and CD33-positive cells from eight patients
with MDS and monosomy 7. The results of this study
suggest that clonality is usually observed in committed
myeloid populations.
MATERIALS AND METHODS
Patients. The clinical characteristics of the patients in this
report are listed in Table 1. All patients had a confirmed diagnosis
of monosomy 7 by standard cytogenetic techniques.13The median
age in this group of patients was 8 years, ranging from 1 year to 65
years. The disease in each patient was classified according to the
morphologic criteria of the French-American-British (FAB) Cooperative Group.14 At the time of sampling, three patients were
diagnosed as having juvenile chronic myelomonocytic leukemia
Table 1. Clinical Characteristics of Eight Patients With Monosomy 7
Patient
Age (vrV
Sex
1
1/M
2
3
4
65lM
35lM
6/M
5
3/M
6
8/F
7
8
14/M
23/M
Diagnosis
Juvenile
CMMoL
RA + AML
RA
Juvenile
CMMoL
RA
Juvenile
CMMoL
Sec. MDS
RAEB AML
-
Treatment Before
Sampling
Unr. LSBMT
Unr. LSBMT
GM-CSF
Chemotherapy
Supportive care
Supportive care
Splenectomy
Chemotherapy
Splenectomy
Chemotherapy
Chemotherapy
Chemotherapy
Abbreviations: M, male; F, female; RA, refractory anemia; RAEB,
refractory anemia with excess of blasts; AML, acute myeloid leukemia;
Sec. MDS, secondary myelodysplastic syndrome; Unr. unrelated;
LSBMT, T-cell-depleted bone marrow transplant using lectin agglutination and €-rosette depletion.
(CMMoL), two patients had refractory anemia, one patient (no.7)
developed secondary MDS 6 years after treatment for Hodglun's
disease, and in two patients the disease had converted to acute
myeloid leukemia.
Patient 1received two T-cell-depleted bone marrow grafts from
an unrelated donor for juvenile chronic myelomonocytic leukemia
(Table 1). Granulocyte-macrophage colony-stimulating factor (GMCSF) was administered because of persisting low peripheral blood
counts after the second transplant. At the time of sampling, the
peripheral blood cells consisted mainly of monocytic cells that were
mostly expressing monosomy 7. Five patients had chemotherapy
before sampling and two patients received supportive care before
sampling.
T- and B-cell lines. To establish T-cell lines, fresh or cryopreserved peripheral blood mononuclear cells (PBMC) or peripheral
blood lymphocytes (PBL) were cultured with irradiated (3,000
rads) third-party PBMC in RPMI-1640 supplemented with penicillin/streptomycin, 15% human serum, 10 kg/mL phytohemagglutinin (PHA-P), and MLA-144 conditioned medium obtained from a
gibbon lymphosarcoma cell line as a source of interleukin-2 (IL-2).
The resulting cell lines were fed twice weekly, once with the above
conditioned medium alone and once with irradiated third-party
feeder cells suspended in the conditioned medium.
B-lymphoblastoid cell lines (BLCL) were established by transformation of lymphocytes with EBV using standard techniques. Fresh
or cryopreserved PBMC or PBL were suspended in a medium
consisting of RPMI-1640 supplemented with penicillin/streptomycin, 15% fetal calf serum (FCS), 25% culture supernatant of the
EBV secreting cell line B95-1, and 1 pg/mL PHA for 24 hours.
Cells were then transferred to 24-well plates in the same medium
minus PHA for the second 24 hours, and fed daily for approximately the next 10 days with RPMI-1640 supplemented with
penicillin/streptomycin, 10% FCS. At that time the cells were
usually transferred to flasks and maintained with thrice weekly
feeding with the medium supplemented with 10% FCS.
Sorting. PBMC were separated from granulocytes and RBCs by
Ficoll-Hypaque (Nycomed Lymphoprep, Accurate Chem. Sci Corp,
Westbury, NY) density gradient and stained with directly conjugated antibodies. The Leu-4 (CD3; Becton Dickinson, San Jose,
CA), Leu-16 (CD20; Becton Dickinson), leu-20 (CD57; Becton
Dickinson), Mol (CDllb; Coulter, Hialeah, FL), My9 (CD33;
Coulter) antibodies were used for these studies. After staining,
cells were either directly used without fixation or fixed in 0.5%
formaldehyde (wt/vol) for 10 minutes. In most cases we avoided
fixing cells with fdrmaldehyde because fixation interferes with the
hybridization resulting in weak fluorescent signals. Cells were
sorted using a FACSTAR PLUS (Becton Dickinson). For the T
(CD3), B (CD20), natural killer (NK) (CD57), and monocytes
(CDllb; dull fluorescent signal) a window was chosen based on
low-side scatter. For granulocytes (brightly fluorescent with Mol/
FITC) and CD33-positive cells no window for scatter was set.
Unfixed cells were sorted directly into an Eppendorf tube filled
with methanol/acetic acid. Fixed cells were sorted directly onto
slides (HTC coated slides; Cell-line) and the slides were subsequently air dried.
Fluorescent in situ hybridization. For methanol/acetic acid (vol/
vol 3:l) fixed cells, the procedure of Pinkel et alls was followed as
described. Briefly, methanol/acetic acid fixed cells were put onto
slides and air dried. Subsequently, slides were put in 2X SSC (1X
SSC is 150 mmol/L NaCl, 15 mmol/L sodium citrate) for 10
minutes at room temperature (RT). Denaturation of the doublestranded DNA was achieved by heating the slides in a solution of
70% formamide in 2X SSC (pH 7.4) for 2 minutes at 70°C.
Thereafter, the slides were dehydrated by 2-minute exposures to
graded ethanol solutions at 4°C. Following this preparation 10 kL
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CLONAL ANALYSIS OF MDS
219
of probe D7Z1 (10 pg/mL biotin-labeled; Oncor, Gaithersburg,
MD) and 10 FL salmon sperm DNA (500 mg/mL; Sigma, St Louis,
MO) or 10 pL human placenta DNA (500 mg/mL; Sigma) was
mixed with 80 pL of a hybridization solution containing 50%
formamide/2X SSC/lO% dextran sulphate (final concentration).
Ten microliters of this hybridization mixture was put on the slides
and covered with a coverslip. Hybridization took place overnight at
37°C. Stringent washing consisted of 3 x 5 minutes in 50%
formamidein 2X SSC at 42"C, followed by 3 x 5 minutes in 2X SSC
at 42°C. Subsequently, cells were washed for 20 minutes at RT in
1xssc.
Cells fixed in formaldehydewere treated according to a modified
protocol described by Durnam et a1.16 After storage in 70%
ethanol, cells were hydrated in phosphate-buffered saline (PBS),
permeabilized in PBS/O.5% Triton, and denaturated at 85°C. The
rest of the procedure was identical to the protocol described above.
Probe detection. After the washing in 1X SSC, slides were
dipped in a solution containing 4X SSC/O.l% "-40 for 3 minutes
followed by 2 x 3 minutes in 4X SSC. The biotinylated probes were
detected by applying 20 pL avidin/FITC (5 pg/mL, in 4X SSC/l%
bovine serum albumin [BSA]; DCS quality; Vector Laboratories
Inc, Burlingame, CA) for 30 minutes. The slides were washed in 4X
SSC/O.l% NP-40,4X SSC, and 4X SSC sequentially for 5 minutes
each. Amplification of the fluorescence signal was achieved by
extra incubation with biotinylated anti-avidin (10 pg/mL; Vector
Laboratories Inc) followed by another layer of avidin/FITC. The
nuclei were counterstained with propidium iodide (1 to 2 Fg/mL;
Sigma) dissolved in antifading s01ution.l~A Nikon or Zeiss
fluorescence microscope were used for visualization. At least 200
cells were scored.
RESULTS
Expression of monosomy 7 in patient cells, Each patient
had documented monosomy 7 by standard cytogenetics. In
most cases karyotype analysis was performed on bone
marrow samples and in all samples the majority of
metaphases showed a deletion of one chromosome 7 (Table
2). The percentage of cells with monosomy 7 ranged from
50% to 100%.
For our study of the lineages affected by MDS, fresh or
thawed peripheral blood cells and/or bone marrow cells
were examined for the expression of monosomy 7 using
FISH analysis. Peripheral blood cells from eight healthy
individuals were used as controls. The number of chromosomes 7 were enumerated by counting the number of bright
fluorescent spots within a nucleus of a cell. The D7Z1 probe
recognizes repetitive sequences on chromosome 7.18 We
used a biotinylated probe and after adding avidin/FITC,
relatively weak fluorescent spots were seen within the
Table 2. Cytogenetic Analysis of Patient Samples
Patient
Material
Chromosomal
Abnormality
% Ex~ression
MetaDhases
BM
BM
BM
BM
BM
BM
BM
PBMC
Monosomy 7
Monosomy 7
Monosomy 7
Monosomy 7
Monosomy 7
Monosomy 7
Monosomy 7
Monosomv 7
100
63
100
100
100
100
50
81
23
24
22
22
21
22
20
21
Abbreviation: BM, bone marrow.
No. of
nuclei. After amplification of the fluorescent signal with
anti-avidin and another layer of avidin/FITC, clear visible
spots were seen.
When salmon sperm DNA was applied as carrier, sometimes multiple spotswere seen indicating nonspecific hybridization. Suppression of nonspecific hybridization was
achieved by using human placenta DNA instead of salmon
sperm DNA.19s20Using this procedure to suppress nonspecific hybridization, two fluorescent spots were seen in 97%
of the cells in control samples. The false-positive rate for
the detection of monosomy 7 was 3% with a standard
deviation of 2% (N = 8). The implication of these values is
that the diagnosis of monosomy 7 in methanol/acetic acid
fixed samples can be made when more than 7%
(mean + 2 x standard deviation) of the cells express only
one chromosome.
Results of the FISH analyses of patients samples are
shown in Fig 1.The expression of monosomy 7 ranged from
26% to 86% in interphase nuclei of peripheral blood
mononuclear cells in six patients. A bone marrow aspirate
and peripheral blood was taken from patient 5 on the same
day. FISH analyses of both samples showed that 26% of the
mononuclear cells in peripheral blood expressed monosomy 7 and 50% of the bone marrow cells. Because there
were not many cells frozen from patient 5, it was decided to
use all peripheral blood cells for establishing T- and B-cell
lines and for two sorting experiments. The diagnosis of
monosomy 7 was made for patient 5 by standard cytogenetic
analysis of a bone marrow aspirate. FISH analysis confirmed the diagnosis of monosomy 7 in a bone marrow
sample of patient 6. Bone marrow cells were examined in
this patient because all peripheral blood cells had to be
used for other experiments. An example of a cell expressing
monosomy 7 and a cell with 2 chromosomes 7 is shown in
Fig 2A.
When looking at the interphase nuclei, granulocytes can
be identified based on the size and shape of the nucleus in
most patients. In the initial studies it was evident that
granulocytes expressed monosomy 7 while cells with a small
round nucleus (presumptively lymphocytes) were diploid
(Fig 2B). These observations suggested the possibility that
monosomy 7 was not expressed in all cell lineages. Therefore, in subsequent studies, T- and B-cell lines were
established and PBMC populations were sorted from each
patient to examine the expression of monosomy 7 in cells of
different lineages.
Expression of monosomy 7 in T- and B-cell lines. When
the T- and B-cell lines were established, their immunophenotype was confirmed by immunofluorescence using an
anti-CD3 antibody as a marker for T cells and an antLCD20
antibody for B cells. From patient 1,samples were obtained
after an allogeneic bone marrow transplantation. The
patient did not engraft after two sex-mismatched bone
marrow transplants (patient 1; see Table 1). Because the
patient was male, cells of host origin could be identified
with a probe for the Y chromosome,
In most cases, cells from the T-cell lines were evaluated
approximately 2 to 3 weeks after initiation of the cultures.
The T-cell line from patient 1 had already been established
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GERRITSEN ET AL
220
UPN
3
3
Material
PBMC
BM
3
3
5
0%
20%
40%
60%
for 6 months at the time of sampling. For all the other
patients we tried to avoid the effect of overgrowth of normal
(diploid) cells by sampling as early as possible after starting
a T-cell line. Every T-cell line examined consisted of 98% to
100% diploid cells. The mean number of cells expressing
monosomy 7 was 1.9% 2 1.1% (Table 3), which is not
significantly different from normal controls (3% k 2%).
An EBV-transformed B-cell line was usually established
after 2 to 4 weeks. Samples were taken to examine the
expressionof monosomy 7 as soon as the lines were growing
80%
100%
Fig 1. Expression of monosomy 7 in peripheral
blood and bone marrow samples used for this study
as determined by fluorescent in situ hybridization.
Cells from patient 5 were unavailable for FISH anaiysir. Qray area, normal range (mean f 2 SD).
steadily in T25 flasks. Similar to the T-cell lines, the vast
majority of the cultured B cells were diploid and only
1.1% 2 1.2% of the cells expressed a single fluorescent spot
consistent with monosomy 7 (Table 3). In the cultures set
up to establish B-cell lines from patients 1and 8, only large,
slowly proliferating cells were observed and these cells
failed to proliferate after about 3 months. Immunophenotypic analysis of these cells showed that 94% of the cells
were Mol (CDl1b)-positive. Of these cells, 94% and 95%
of the cells expressed monosomy 7. These results indicate
fi
A
Fig 2. (A) Example of monosomy 7 and diploidy in interphase cells detected by fluorescent in situ hybridization (patient 1). (8)Monosomy 7
expression in a granulocyte, while the cell with a round nucleus (presumablya lymphocyte) is diploid (patient 3). (C)Detection of chromosome 7 in
a metaphasecell with the probe D7Z1 (T-cell line patient 5).
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CLONAL ANALYSIS OF MDS
221
Table 3. Expressionof Monosomy7 in 1-and B-Cell Lines as
Determined by FISH
Patient
T-cellLine (%)
6-Cell Line (%)
0.5
3.0
2.7
2.9
2.0
2.0
0.0
3.0
0.5
1.9
2.9
1.o
0.0
NA
0.0
t
1.9
1.1
Abbreviation: NA, not available due to contamination.
*In another attempt no B-cell line was established, but cells were
growing that were expressing only one chromosome 7.
tNo B-cell line was established. Cells were growing that were
expressingmonosomy 7.
that it is important to rule out any contamination of tumor
cells when analyzing B-cell lines for the expression of
abnormal chromosomes in myelodysplasticpatients.
Our analysis of the T- and B-cell lines strongly suggested
that the T- and B-cell lineage were not involved in this
disease. Nevertheless, we observed one fluorescent spot in
some cells. This observation could be explained in several
ways. For example, our observations through the fluorescent microscope are two dimensional, while the cell structure is three dimensional. Hence, it is possible for one
fluorescent spot in a nucleus to be overlapped by another
single fluorescent spot in the same nucleus. It is also
possible that cells with one spot actually contain two
chromosomes 7 but FISH in certain cases is unable to
detect both chromosomes. Alternatively, these cells with
one fluorescent spot actually contain one chromosome 7
and are affected by the disease, in which case even if 1% of
the cells had only one chromosome 7, the disease could be
affecting cells in the T- and B-cell lineages.
We prepared metaphase cells from six T-cell lines and
scored the number of chromosomes 7 with the aid of
fluorescent probes and standard cytogenetic techniques
(Table 4). In none of these samples was a deletion of
chromosome 7 detected. These results further support our
initial contension that the T-cell lines are diploid. Unfortunately, natural selection in favor of diploid cells cannot be
ruled out when T- and B-cell lines are established. Therefore, we developed a procedure to study the expression of
Table 4. Expressionof Monosomy7 in T-cell Lines as Determined by
Standard Cytogenetics and Fluorescent In Situ Hybridization
Cytogenetics
FISH
No. of
Patient
1
2
3
4
5
6
Control
% Monosomy 7
0
0
0
0
0
Metaphases
0
10
15
9
14
5
No. of
% Monosomy 7
Metaphases
0
0
0
0
0
0
0
32
37
40
62
43
19
monosomy 7 in populations of lymphoid and myeloid cells
isolated by sorting directly from the blood or marrow.
Expression of monosomy 7 in sorted cell populations. For
this part of the study we stained cells with monoclonal
antibodies, sorted the cells according to their scatter
pattern and fluorescence signal, and subsequently used the
FISH procedure to visualize the number of chromosomes 7
in the sorted subpopulations. In our initial experiments we
compared two different procedures: (1) fixation of the
stained cells in 0.5% formaldehyde followed by sorting onto
slides and subsequently storing the slides in 70% ethanol;
and (2) no fixation with formaldehyde, sorting directly into
Eppendorf tubes filled with methanol/acetic acid. Although cell morphology was best maintained when cells
were fixed in formaldehyde, FISH analysis was suboptimal
and required additional amplification of the fluorescent
signal. The best FISH results were seen without the fixation
with formaldehyde. The choice of method in our studies
depended on the availability of the cell sorter on the same
day that we received patient samples. In three control
samples the rate of false-positive results ranged from 4.3%
to 7.2%with both methods.
After sorting T cells (CD3) obtained from normal controls, 4.8% f 4.4% of the cells had only one fluorescent
spot. In our eight patients the expression of monosomy 7 in
the T cells did not differ from the control values (Fig 3). B
cells could be purified from five patients. An average of
5.4% of these purified B cells displayed one fluorescent
spot in the nucleus (Fig 3), which is clearly within the
normal range (controls: 4% 2 2%). B cells could not be
purified by the cell sorter in three of eight patients because
in two patients (nos. 1 and 2) no B cells could be detected
and in the other patient (no. 5) the number of cells did not
allow sorting of all subpopulations. NK cells were purified
by selecting cells that expressed the CD57 antigen and had
an intermediate forward scatter signal and a low-side
scatter signal. In three patients (nos. 2,4,8) less than 1% of
the peripheral blood cells were NK cells and for one patient
(no. 5) not enough cells were available to study NK cells.
For the four patients studied, the NK cells expressed one
fluorescent spot in a mean of 6.3% of the cells (controls:
5% k 4%). In summary, cells in the lymphoid cell lineage
(T, B, and NK cells) in patients with MDS did not differ
significantly from the control values in the proportion of
cells with a single fluorescent signal, confirming that the
lymphoid cell lineage is not involved in MDS associated
with monosomy 7.
Three myeloid subpopulationswere distinguished according to their expression of the CDllb antigen and the CD33
antigen. The cells with a low expression of the CDllb
antigen had a low-side scatter signal and the morphologic
features of monocytes. In seven of eight cases we were able
to isolate this subpopulation. In contrast to the results in
the lymphoid populations, this myeloid subpopulation exhibited monosomy 7 in the majority of patients. In six of seven
patients the percentage of cells with monosomy 7 differed
significantly from the control values (6% 2%), ranging
from 18% to 78% (Fig 3). Cells with a high expression of the
CDllb antigen usually had a high-side scatter signal and
*
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GERRITSEN ET AL
222
Lymphoid lineage
/
UPN
I
CD3
CD20
I
Myeloid lineage
E
CD57
CDllb
=
\
CD33
CD11b
9*2%
na
61 f 2%
26&24%
43&14%
na
21%
43%
80%
low
I
hlah
1.
5*=
na
7%
2
4i=
ne
na
3.
5&3%
6%
8&5%
4.
3&1%
5%
n
5.
7%
ne
na
19%
na
ne
6.
4&1%
6%
3%
58&14%
47%
81 & W
7.
5%
3%
7%
18%
20%
23%
8.
4&1%
7%
na
78&6%
89&W
91&2%
Control
5&4%
6&2%
7&3%
4&2%
a
524%
;
:
:
*
90&6%
either the appearance of granulocytes (no. 3) or blasts (no.
1 and 8) based on the size and shape of the nuclei. In all the
five patients, where we were able to isolate this myeloid
subpopulation, the percentage of cells expressing monosomy 7 differed significantly from control values (5% -C 3%;
Fig 3). The majority of the cells (range 61% to 91%)
exhibited one fluorescent spot in four of these five patients,
and in patient 7 23% of the cells expressed monosomy 7.
We also distinguished myeloid populations on the basis of
expression of the CD33 antigen. The myeloid subpopulation identified by the anti-CD33 antibody contains a variety
of cells ranging from mature monocytes to the common
progenitor for monocytes and granulocytes. Again, CD33positive cells from each of the six patients evaluated
contained a significant proportion of monosomy 7 cells
ranging from 20% to 90% (Fig 3). These results indicate
that cells in the myeloid lineage bear monosomy 7 as a
marker of the disease.
DISCUSSION
In this study, we have focused on the expression of
monosomy 7 in the lymphoid and myeloid subpopulations.
We used the FISH technique because it is unique in its
ability to visualize the chromosomes 7 within a single
nucleus using a chromosome-specific probe. In addition,
this technique is applicable to all patients and it is not
dependent on whether patients show any heterozygosity for
Y-linked or autosomal loci. Thirdly, the technique of FISH
5k3%
Flg 3. Expmrlon of monosomy 7 incell populations, which
were sorted according to the expression of sudace antibodies
5pecMc for T cells (CD3). B cells
(CD20). NK cells (CD57). monocytes (CDllb), granulocytes
(CDllb). and myeloid progenitors (CD33). no, Not available
due to either no cells present or
not enough cells to separate all
the six cell subpopulations; no
scoring possible because of background fluorescence.
.,
is sensitive enough to distinguish between subpopulations
that do or do not express monosomy 7. In our hands the rate
of false positives was 3% f 2% for unseparated cells and
7% f 3% was the highest rate detected in purified
subpopulations. Two observations might explain why a
higher frequency of false-positive cells was observed after
sorting cells: (1) After drying, the size of the cells is smaller
after sorting in PBS solution onto slides, which means that
the chance of juxtapositioning of the two hybridization
regions is higher. When the distance between two signals is
not large enough, two signals will be scored as one signal.
(2) The very bright fluorescent antibodies bound to the cell
surface did cause some background. This background
staining hampered an accurate scoring. However, the sensitivity was similar to that reported by Anastasi et a12' for the
detection of monosomy 9 with a false-positive rate of
7.8% f 1.6%. Nevertheless, despite the higher frequencyof
false positivity in purified subpopulations, the sensitivity
appeared to be good enough to detect significant differences in the proportion of cells expressing monosomy 7 in
lymphoid and myeloid subpopulations.
Cytogenetic analysis of metaphase cells from bone marr o w samples submitted at the time of clinical diagnosis
confirmed the presence of monosomy 7 in the majority of
dividing cells. Analysis of interphase cells with FISH
confirmed that every patient also had peripheral blood cells
expressing monosomy 7. Although cytogenetic analysis of
bone marrow samples detected only monosomy 7 cells in
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CLONAL ANALYSIS OF MDS
five of eight patients, this may only reflect the fact that
myeloid and erythroid progenitors constitute the vast majority of actively dividing cells which would be detected in the
bone marrow. The FISH analysis of peripheral blood
samples showed that only a proportion of the cells expressed monosomy 7. The sorting experiments further
confirmed that there is a mixture of normal cells and cells
expressing monosomy 7 in peripheral blood.
Monosomy 7 was not expressed in cells from T-cell lines
or in purified T cells as determined by FISH in our study.
These results are concordant with data published by Kere
et al.12 In their study, peripheral blood lymphocytes were
purified by Ficoll-Hypaque gradient and the expression of
monosomy 7 was studied by looking at the polymorphic
expression of the MET-proto-oncogene and the arginosuccinate synthetase pseudogene. Both genes are located on
chromosome 7. In five patients with MDS associated with
monosomy 7, none of the lymphocyte fractions studied
expressed monosomy 7. Lawrence et a17 found similar
results when they studied metaphases, obtained after PHA
stimulation, in two patients with MDS associated with a 13q
deletion. In contrast to these studies and our own study, two
other studies about the clonal origin of MDS concluded
that the T cells may be affected by MDS. Janssen et a19used
polymerase chain reaction (PCR) to detect point mutations
in the Ki-ras and N-ras oncogenes in purified subpopulations. They detected point mutations in T cells from two
patients with chronic myelomonocytic l e ~ k e m i aHowever,
.~
it is possible that contamination could have attributed to
these results because the purified T cells contained 5% to
10% contaminating myeloid cells. Tefferi et all0 have
reported that T cells from four patients with MDS showed a
skewed pattern using RFLP-methylation analysis, suggesting that the T cells were affected by MDS.
The reports about the involvement of the B cells in MDS
are equivocal. In two patients with a 13q deletion, reported
by Lawrence et al,’ karyotyping of B-cell lines showed that
3% and 13% of the metaphases had a deletion in the long
arm of chromosome 13. In another patient with MDS
associated with distinctive chromosomal abnormalities ( l l q
and trisomy B), none of 16 B-cell lines evaluated possessed
these abnormal karyotypic features. However, an analysis
of the B-cell lines from this same female patient who was
heterozygousfor the enzyme glucosed-phosphate dehydrogenase (G6PD) showed that 21 of 24 lines were expressing
type B G6PD strongly suggesting that the growth of these B
cells was at least skewed.8 Our analyses of B-cell lines as
well as the B cells (CD20) isolated from the peripheral
blood were concordant, and indicated that these B-cell
populations are not affected by monosomy 7. A potential
source of confusion in studies of lymphoid cell lines from
patients with MDS was illustrated in two cases in our series
from whom we tried to establish B-cell lines. In these cases
only Mol+ tumor cells grew in the cultures. These cells also
expressed only one chromosome 7. In both instances the
peripheral blood cells of the patients also expressed monosomy 7 in more than 80% of the cells both by karyotyping
and FISH. These findings suggest that observations of
223
chromosomal abnormalities in lymphoid lines could, in
some instances, be attributable to contamination with
diseased monocytic cells expressing monosomy 7.
All studies about the clonal origin of MDS indicate that
neutrophils and monocytes are affected by the disease. Our
studies of purified myeloid subpopulations also demonstrate that monosomy 7 is predominantly expressed in
myeloid cells. Furthermore, our analyses of sorted myeloid
populations also show that the cells with monosomy 7 can
differentiate into granulocytes and monocytes. Kere et all2
reported identical results. This latter study and our study
about the clonal origin of cells with monosomy 7 strongly
suggest that MDS associated with monosomy 7 is a disease
of committed progenitor cells without the loss of their
potential to differentiate into monocytes and granulocytes.
Our study and that of Kere et all2 cannot exclude the
possibility that the cells in the lymphoid lineage express
monosomy 7, because the techniques used are not sensitive
enough to detect the expression of monosomy 7 in less than
3% of the subpopulations. For example, in our study, 98%
of the nuclei of the T-cell lines derived from patients with
MDS had 2 chromosomes 7 as assessed by FISH analysis, a
result which does not differ from that obtained when T cells
from normal individuals are evaluated. Because it was
desirable to investigate whether the 2% cells did or did not
express monosomy 7, metaphases were prepared from the
T-cell lines. All metaphases had two chromosomes 7.
However, the number of metaphases evaluated was again
not sufficient to exclude the possibility that 1% of the cells
express monosomy 7. At least 300 metaphases have to be
analyzed to accept with 95% confidence limit that 0% of the
metaphases do not express monosomy 7.” Alternative
approaches, such as RFLP or probes for distinctive mutations on chromosome 7, could be proposed to address this
issue. However, these techniques may also yield ambiguous
results because they cannot distinguish between lymphoid
cells and cells contaminating the sorted cell fractions.
Our patient population with MDS is rather young with a
median age of 8 years, while the majority of the patients
with MDS are over 60 years old? One study has suggested
that there are two different forms of acute nonlymphocytic
leukemia. Leukemias mainly involving the granulocytic cell
lineage, in which normal early progenitor cells were identified, have been reported in children and young adults. In
contrast, older patients have another type of acute nonlymphocytic leukemia in which clonal features are detected in
early progenitors of all lineagesu Because we performed
our studies in a relatively young group of patients, the
possibility exists that a different pattern of lineage involvement might be in a group of older patients. However, the
one patient over 60 years old who was included in our study
showed no involvement of the lymphoid lineage, which is in
accordance with our observations in younger patients.
This study about the clonality of MDS associated with
monosomy 7 shows that monosomy 7 affects predominantly
the myeloid cell lineages. Evaluations of T- and B-cell lines
as well as purified lymphoid subpopulations strongly suggest that the lymphoid cells are not expressing monosomy 7.
From www.bloodjournal.org by guest on March 5, 2016. For personal use only.
GERRITSEN ET AL
224
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1992 80: 217-224
Clonal analysis of myelodysplastic syndrome: monosomy 7 is
expressed in the myeloid lineage, but not in the lymphoid lineage as
detected by fluorescent in situ hybridization
WR Gerritsen, J Donohue, J Bauman, SC Jhanwar, NA Kernan, H Castro-Malaspina, RJ O'Reilly
and JH Bourhis
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