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Evidence for Nonclonal Hematopoietic Progenitor Cell Populations in Bone
Marrow of Patients With Myelodysplastic Syndromes
By Haruhiko Asano, Haruhiko Ohashi, Masatoshi Ichihara, Tomohiro Kinoshita, Takashi Murate,
Miya Kobayashi,
Hidehiko Saito. and Tomomitsu Hotta
Clonality of marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDSI wasanalyzed by X-chromosome inactivation pattern using polymerase chain reaction
(PCR). Fivefemale patients were included in this study; two
with refractory anemia (RA) and three with RA with excess
blasts (RAEBI. They were heterozygous for BstXl restriction
fragment length polymorphisms (RFLP)of the X-chromosome-linked phosphoglycerate kinase (PGK) gene. In each
patient, erythroid and nonerythroid colonies, grown in the
presence of erythropoietin and granulocyte-macrophage
colony-stimulating factor (GM-CSFI, exhibited no remarkable difference in clonal constitution. Two patients showed
only one methylation pattern, suggesting the monoclonal
origin of hematopoietic progenitor cells.Coloniesof two
other patients exhibited predominant and minor methylation patterns in PGKgene, indicating that nonclonal progenitor cells remain a minor population. The bone marrow of
one patient appeared to contain a greater proportion of nonclonal progenitors. Stem cell factor (SCF), a potent colony
stimulating factor, enhanced both erythroid and nonerythroid colony formation. However, it did not notably alter
the clonal constitutions. We conclude that nonclonal hematopoietic progenitor cells canpersist in a substantial number
of MDS patients.
0 1994 by The American Society of Hematology.
T
applicability forclonal studies, evenfor patients with no
genetic markers. Using the method, many investigators disclosed the clonal nature of MDS,".''~?'' as well as other neoplastic
Because
X-inactivation
the pattern
is
sustained in the daughter cells in a highly stable manner,'h
clonal analysis of individual colonies grown in culture
should show the clonality of hematopoietic progenitor cells.
Inclusion of polymerase chain reaction (PCR) in the genetic
analysis has made it possible to examine the clonality of a
small cell population.?* In this study, we wish to clarify the
clonalorigin
of MDS marrowhematopoieticprogenitor
cells. X-inactivation patterns of single erythroid (burst-forming units-erythroid[BFU-E])
and nonerythroid colonies
wereanalyzedusing
PCR. Resultssuggestthatnonclonal
progenitor cells remain in a substantial number of MDS
patients, which might be important implication for the therapy of MDS.
HEMYELODYSPLASTICsyndromes(MDS)are
a
morphologically defined heterogeneous group of stem
cell disorders characterized by maturational defects of marrow hematopoietic cells, resulting in peripheral blood cytopenias.'.? Clonal hematopoiesis isanothercharacteristicin
MDS.' Analyses using X-linked isoenzyme system of glucose-6-phosphate dehydrogenase (G6PD)
revealed the clonal
origin of peripheral blood cells of MDS patients."' These
studies have provided valuable information on the clonality
ofhematologicdisorders
because itisapplicable
for the
analysis of erythrocytes and platelets lacking nuclei. However, the rarity of females with heterogeneity of G6PD has
limited further study. Cytogenetic analyse^"'^ and studies of
nonspecific gene rnutation~,'~
further clarifyingthe clonal
nature of MDS, have been powerful tools for direct demonstration of the clonal population. If the karyotypic (genetic)
changes occurred after the clonal evolution, the dataof such
analyses might not reflect the real clonality in the patient.
For thatreason,reportsonthemosaicism
of normal and
abnormal karyotypes in hematopoietic progenitor cells"'.' I
do not necessarily provide evidence for the presence of normal clone in MDS. In any event, investigators have targeted
mainly mature peripheralblood cellsfor clonalanalysis.
Adequate information on the clonality of marrow progenitor
cells has not been available in MDS.
Vogelstein et al" established an elegant method for clonality analysis by means of DNA polymorphisms atX-linked
loci, which uses random X-chromosome inactivation in females." This strategyhas the great advantage of general
From the First Department of Internal Medicine, and the Second
Department of Anatomy, Nagoyu Universiv School of Medicine,
Nagoya, Japan.
Submitted December 2, 1993; accepted March 23, 1994.
Address reprint requests to Haruhiko Asano, MD, the First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoyu 466, Japan.
The publication costs of this article were defrayed
in part by page
chargepayment. This article must thereforebeherebymarked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8402-0006$3.00/0
588
MATERIALS AND METHODS
Patients. Five female patients with primary MDS were included
in this investigation (Table 1); two with refractory anemia (RA) and
three with RA with excess blasts (RAEB) according to the FrenchAmerican-British(FAB) nomenclature.'' Theywereheterozygous
for BstXI restriction fragment length polymorphisms (RFLP) of the
X-chromosome gene phosphoglycerate kinase ( PG K ) and received
neither chemotherapy nor bone marrow transplantation before the
study. Their marrow and peripheral
blood samples were obtained
with informed consent. As a control, two healthy female volunteers
whowereheterozygousforthe
RFLP were also included in this
study, after informed consent was obtained.
Cell preparation. Light-density mononuclear cells (MNC) were
obtained from marrow and peripheralblood samples by Ficoll-Paque
(Pharmacia, Uppsala, Sweden) density gradient centrifugation. Marrow MNC were washed and resuspended in RPM1 1640 (GIBCO,
GrandIsland,NY)
with 20% fetal calf serum(FCS,Boehringer
Mannheim, Mannheim, Germany) at I X IOh/mL and incubated for
2 hrs at 37°C in a fully humidified atmosphere of 5 % CO,, then
nonadherent cells were collected. Polymorphonuclear cells (PMN)
were recovered from the peripheral blood erythrocyte
pellet by removal of lysed red cells after incubation in Tris.NH,CI buffer. The
purity of the PMN obtained was 94.0% 2 3.5% (mean t SD) by
morphologicexamination. In somepatients,Tlymphocytes
were
separated from peripheral blood MNC by agglutination with sheep
Blood, Vol 84, NO 2 (July 15). 1994: pp 588-594
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EMATOPOIETIC NONCLONAL
589
IN MDS
Table 1. Clinical and Hematologic Data of Patients at theTime of Study
Peripheral Blood
UPN
1
2
3
4
5
AgeISex
46/F
71F
71F
75F
68/F
Diagnosis*
RA
RA
2.9 RAEB
RAEB
RAEB
Time After
Diagnosis
(mo)
13
9
26
66
13
Bone Marrow
WBC
c1oSn)
2.5
3.3
Blast
1%)
0
0
2
3.8
1.1
2
0
12.7
Hb
WdL)
Platelets
12.1
7.1
8.7
6.6
4.7
9
6
( 109/u
42
99
43
Blast (96)
<l
<l
4
8
Karyotype
46,XX
46,XX
46,XX
46,XX
46,XX
Abbreviations: UPN, unique patient number; Hb, hemoglobin.
* French-American-British(FAB) classification.
redblood cells (Kyokuto, Tokyo, Japan). Immunohistochemical
analysis showed 93.7% 2 1.5% of the cells obtained were CD2+.
Hematopoietic growth factors. Recombinant human erythropoietin (EPO) and stem cell factor (SCF) were generous gifts from
Kirin Brewery CO(Tokyo, Japan). They were used at a concentration
of 100 ng/mL and 4 UlmL, respectively. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 ng/
mL) was a generous gift of Sumitomo Pharmaceutical (Hyogo, Japan).
Cell culture. Cells from MDS and control marrow samples were
grown at 5 x l@/mL in 1 mL of Minimum Essential Medium-a
(MEM-a, GIBCO) containing 1.1% methylcellulose supplemented
with 20% FCS, 1% dialyzed bovine serum albumin (BSA; Sigma
m o m of 2-mercaptoethanol
Chemical CO, St Louis, MO), 5 x
(2-ME; Nacalai Tesque, Kyoto, Japan), EPO, and GM-CSF, either
with or without SCF in a 35-mm culture dish (Nunc, Naperville, Ill)
for 14 days at 37°C in a fully humidified atmosphere of 5% CO2.
After 14 days of incubation, cell groups composed of more than 40
cells were scored as colonies.
DNA extraction and processing. Individual red-colored (erythroid) and other (nonerythroid) colonies were randomly plucked by
hand with a micropipette and immediately placed in 200 pL buffer
containing 1% sodium dodecyl sulfate (SDS), 2 mmol/L EDTA,
20 pmoVL Tris (pH 7.4), and 1 mg/mL proteinase K (Boehringer
Mannheim) followed by incubation for 12 hours at 37°C. Highmolecular weight DNA, extracted from each colony with phenol/
chloroform24using 44 pg of glycogen (Boehringer Mannheim) as a
DNA container, was equally divided into two aliquots; one was
digested by 40 U of HpaII, methylation-sensitive restriction endonuclease (all restriction enzymes except BstXI were purchased from
Boehringer Mannheim) for more than 6 hours at 37T, and the
other was left undigested. Similarly, high-molecular weight DNA
of blood T lymphocytes or MNC and PMN was extracted and two
aliquots containing about 100 ng of DNA were obtained. One was
digested by HpaII in the same condition as described earlier. The
DNA after HpaII-cutting was precipitated in ethanol and the pellet
was then rinsed once with 70% ethanol.
PCR analysis of PGK gene. Clonality analysis was conducted
according to the method established by Gilliland et alZZwith some
modifications. DNA from each colony and peripheral blood samples
both with and without HpaII digestion were added to 50 pL of a
solution (16.6 mmoVL ammonium sulfate, 67 mmol/L Tris.HC1 [pH
8.8 at25"C],6.7 mmoVL MgClZ, 10 mmoVL 2-ME, 6.7 pmoVL
EDTA, 1.5 mmoVL dNTPs, and BSA at 170 pg/mL) with a set of
outer primers at 1 pmoVL each and 1 U of Taq polymerase (Boehringer Mannheim). Samples were amplified for 35 cycles (1 minute,
94°C; 1 minute, 55°C; 2 minutes, 72°C). One tenth of each product
was further amplified for 50 cycles in the same solution with a set
of inner primers. Sequences of the two sets of primers were described
elsewhere.22Following the amplification, each product was digested
by 40 U of BstXI (New England Biolabs, Beverly, MA) at 55°C for
more than 6 hours, then electrophoresed in 2% agarose geland
stained with ethidium bromide.
Determination of clonal pattern of individual colonies. The
clonal origin of each colony was determined on the basis of the
band pattern of amplified DNA with precutting by HpaII. To assess
the band pattern, artificial cell-mixtures were prepared as follows.
Blood mononuclear cells were obtained from two healthy male volunteers with informed consent; one had PGK gene lacking the BstXI
site, and the other, containing the site. Cells were mixed at the ratios
of 1W0, 95:5, 90:10, 85:15, 80:20, 70:30, 60:40, 50:50, 40:60,
30:70, 20:80, 15:85, 10:90, 5:95, and 0:100, respectively. DNA extracted from each cell-mixture was amplified and then electrophoresed in the same condition as described earlier (Fig 1).
The band pattern of amplified DNA from each colony, with precutting by HpaII, was compared with bands in the artificial cellmixtures. Band patterns between 40:60 and 60:40 were regarded as
ambiguous, and the others as having the inactivated allele without
BstXI site (pattern A, dominant band at 530 bp) or withthe site
(pattern B, dominant band at 433 bp).
The complete digestion with HpaII and BstXl was confirmed
using the male DNA containing the BstXI site. Each step of DNA
digestion and the amplification was performed in the same condition
as noted above. The amplification of the DNA cut with HpaII failed
(not shown), indicating the complete digestion withthe enzyme.
BstXI digestion of products from the DNA undigested with HpaII
yielded a single 433-bp band as in the first lane from the left in Fig
1, which showed that complete digestion with BstXI was achieved.
Clonal analysis of blood PMN by Southern blotting. According
20 pg DNAwas
tothemethod
established by Vogelstein et
completely digested with BgA, BgAI, and EcoRI, then equally divided into two aliquots; one was further cleaved with HpaII, and the
other was left undigested. Each aliquot of DNA was electrophoresed
through 1.5% agarose gel, transferred to a nylon membrane, and
hybridized with a 0.8-kbp genomic probe containing the 5' region
of the PGK gene (provided by Dr J. Singer-Sam, Beckman Research
Institute of the City of Hope, Duarte, CA).
RESULTS
Clonality of erythroid progenitors. In the presence of
EPO and GM-CSF, erythroid (BFU-E) colonies were formed
in all five MDS patients, as well as two control individuals
(Fig 2A), and were randomly plucked. Although each colony
from patients was generally small-sized, DNAwas effectively amplified by PCR (data not shown). The clonal origin
could be determined even if the colony was composed of
less than 100 cells.
In two patients (UPN 3 and 4), all the erythroid colonies
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ASANO ET AL
590
Fig 1. Band patterns of PGK
gene from artificial cell-mixtures
for determining the clonal origin
of individual colonies. Because
of
the
formation
of
heteroduplex,a the intensity of upper
bands is high.
-
YO cells containing theBstXl site
examined had the same X-inactivation pattern (Table 2).
yielding a dominant band at 530 bp (pattern A) as illustrated
in Fig 3A. Colonies of two other patients (UPN 1 and 2)
had extremely skewed inactivation patterns; most of them
exhibited pattern A, while four of 25 colonies in UPN 1 and
two of 22 in UPN 2 produced a dominant band at 433 bp
(pattern B). In UPN 5 , five of 18 colonies examined were
pattern A and other 13, pattern B (Table 2 and Fig 3B). In
control individuals (Cl and C2), the ratios of pattern A to
B of the colonies were 8: 1 I and 8:21, respectively (Table
2). Representative band patterns in C2 are shown in Fig 3C.
Clonality of nonerythroid progenitors. Nonerythroid
colonies grown in the presence of EPO and GM-CSF (Fig
2B) were randomly plucked andtheir clonal origin was determined. In patients UPN 3 and 4, all of the nonerythroid
colonies had only one inactivation pattern (Table 2 and Fig
4A). The colonies of UPN I and 2 gave three band patterns:
pattern A, pattern B, and ambiguous. The ratios were skewed
B
A
T
T
1
2
3
4
UPN
5 C l C 2
1
2
3
4
5 C l C 2
UPN
Fig 2. Erythroid (A) and nonerythroid(B) colony growthin each patient and normal
individual. Adherent cell-depleted marrow mononuclear
cells were stimulated by bothEPO and GM-CSF, and either(W) with or (0)
without SCF. Data represent mean colony number grown from
lo5
mononuclear cells. The error bars depict1 SD from the mean.
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MATOPOIETIC NONCLONAL
IN MDS
591
Table 2. X-Chromosome Inactivation Pattern of Individual Colonies
Pattern of BstXl RFLP
Erythroid
FAB
Nonerythroid
SCF'
A
B
Ambiguous
A
-
-
7
+
-
20
5
+
7
Control ( C l )
-
8
5
Control (C2)
-
4
5
2
2
0
0
0
0
13
11
11
14
21
23
0
+
21
25
20
23
13
17
15
18
15
16
18
21
15
13
4
4
10
6
10
9
UPN
RA
l
+
RA
2
-
+
3
RAEB
4
RAEB
RAEB
5
-
+
+
a
7
0
1
0
0
0
0
0
0
0
0
0
0
0
B
Ambiguous
1
2
3
2
0
1
0
2
2
0
0
0
0
1
13
17
5
13
16
19
0
0
l
1
4
1
4
1
Clonality analysis of erythroid and nonerythroid colonies grown in the presence of both EPO and GM-CSF, and either with or without SCF.
The clonal origin of each colony was determined on the basis of the ratio of amplified DNA. The ratio of between 40:60 and 60:40 in artificial
cell-mixtures (Fig 1)was regarded as ambiguous, and the others as having the inactivated allele without the BstXl site (pattern A) or with the
site (pattern B). The ambiguous pattern may result from contamination by cells having another clonality. The diffuse nature of the nonerythroid
colonies presumably leads to the plucking of cells with a differentclonal origin.
x Symbols
and - represent the stimulation with and without the factor, respectively.
+
as 15: 1: 1 (UPN 1) and 1 5 3 2 (UPN 2) (Table 2). In UPN
5 , the ratio was also skewed as 4: 13:1 (Table 2 and Fig 49).
In controls, three band patterns were observed (Table 2 and
Fig 4C). The clonal constitutions of nonerythroid colonies
of all patients and control individuals were similar to those
of erythroid.
Efects (.f SCF on progenitor cell clortaliv i n MDS. SCF
was added to the culture system to investigate whether it
could alter the clonal constitution of colonies in MDS.
Among patients, colonies of both erythroid and nonerythroid
lineages grew approximately two to six times more in the
presence of SCF (Fig 2AB), which was observed also in two
control individuals. The clonal origin was determined for
colonies plucked randomly(Table 2). In patients and controls
alike, the clonal constitutions of both erythroid and nonerythroid colonies were similar to those grown without SCF.
However, one of 14 nonerythroid colonies in UPN 4 carried
a different pattern of X-inactivation (Table 2). indicating the
presence of nonclonal hematopoietic progenitor cells in the
marrow.
Clonal acquisition ann!\:ed t>v peripheral blood cells. It
is necessary to know if the clonal nature of MDS marrow
hematopoietic progenitor cells was theresult of acquired
clonal evolution or of extreme lyonization. Clonality of peripheral blood PMN and either T lymphocytes or MNC was
analyzed by means of PCR, andtheresults are shown in
Table 3 and Fig S. In PMN of three patients (UPN I , 3 and
4). DNA precut with HpaII produced only an upper (530bp) band, indicating PMN were of monoclonal origin. The
T-lymphocyte DNA gave rise to extremelyskewed (oligoclonal) band patterns in UPN I and 3, which were composed of dominant upper (530-bp) and minor lower (433bp) bands, suggesting that their T lymphocytes included a
smallnonclonal population. Such a difference in clonality
of PMN and T lymphocytes indicated that acquired clonal
events were going on in their hematopoietic system. In UPN
4, the T-lymphocyte DNA also yielded only the upper band,
indicating monoclonal origin. The X-inactivation pattern of
PMN from UPN2 was oligoclonal, but the MNCDNA
produced an unskewed polyclonal band pattern, which im-
Fig 3. X-chromosome inactivation pattern of individual erythroid colonies. Representative band patterns are illustrated. All bands in H(+)
lanes were derived from
DNA precut with Hpall. (A) AnRAEB patient (UPN 3). DNA in H(+) lanes yielded a band at
530 bp, indicating inactivated
X-chromosomes of cells in the colonies lacked BstXl site of PGK gene. (B) An RAEB patient (UPN 5).A dominant band at433 bp isobserved
in each H(+) lane, indicating inactivated X-chromosomes contained the BstXlsite. (C) A normal individual (Cl). Both patterns are noticed.
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ASANO ETAL
592
C
B
A
*
Fig 4. X-inactivation pattern of individual nonerythroid colonies. Representative band patterns are illustrated. All bands in H(+) lanes were
derived from DNA precut with Hpall. (A) An RAEB patient (UPN 4). DNA in H(+) lanes yielded a band at 530 bp. (B) An RAEB patient (UPN 5).
A dominant band at 433 bp is observed in each H(+) lane. (C) A normal individual (C21. Three band patterns are noticed. ‘Denotes ambiguous
band pattern. A dominant 433-bp band in each H(+) lane is often accompanied by a faint 530-bp band, which may be explained either by the
contamination of nonclonal cells or by the escape from restriction endonuclease digestion. Slight contamination of alleles containing the BsrXl
site by the site-lacking alleles might yield the 530-bp band, which was strengthened by the formation of heteroduplex.=
plied that acquired clonal evolution in hematopoiesis took
place in the marrow. UPN 5 , who had a greater proportion
of nonclonal marrow hematopoietic progenitor cells than any
other patients, showed almost the same polyclonal patterns
in PMN and MNC.
Thus, despite the presence of nonclonal hematopoietic
progenitor cells, peripheral bloodPMNofUPN
1 and 2
showed monoclonal and oligoclonal appearances, respectively. To clarify this discrepancy, we further performed
clonal analysis of PMN of these patients by Southern blotting. PMN of UPN 1 appeared to be monoclonal and those
of UPN 2 had an extremely skewed clonal constitution (Fig
6), which were similar to the results of PCR-based clonal
analysis.
DISCUSSION
To know the clonal constitution of MDS marrow hematopoietic progenitor cells, we performed a PCR-based clonal
analysis for colonies grown in culture. X-chromosome inactivation patterns were determined on individual erythroid and
nonerythroid colonies. Nonclonal hematopoietic progenitor
cells were present in four out of five patients tested. In patients UPN 3 and 4, few, if any, nonclonal progenitor cells
were detected; in UPN 1 and 2, nonclonal cells formed a
small progenitor cell population, while UPN 5 had more of
these cells. These clonal constitutions of marrow hematopoietic progenitor cells were well reflected in the clonality of
circulating PMN. PMN exhibited monoclonal or extremely
skewed (oligoclonal) band patterns in UPN I , 2, 3, and 4,
Table 3. Clonal Patterns of Peripheral Blood Cells
Cell Population
UPN
FAB
PMN
T Cells
MNC
1
RA
RA
RAEB
RAEB
RAEB
M
0
-
0
-
P
M
M
P
0
M
-
-
P
2
3
4
5
-
PCR-based clonality analysis was performed on peripheral blood
PMN and either T cells or MNC.
Abbreviations: M, monoclonal band pattern; 0, oligoclonal band
pattern; P, polyclonal band pattern; -, not available.
while PMN of UPN 5 exhibited a polyclonal band pattern.
Itisworth noting that, despite the presence of nonclonal
marrow progenitors, PMN of UPN 1 showed a monoclonal
band pattern. This apparent discrepancy could be caused by
the sensitivity of our PCR-based clonal analysis or by selective release of monoclonal cells into the peripheral blood.
Specifically, the amplification ofDNA from artificial cellmixtures containing 85% cells whose X-chromosomes
lacked the BstXl site apparently yielded only one bandat
530 bp (Fig l).It isvery probable that if the nonclonal
hematopoietic progenitor cell population is small, the circulating PMN show a monoclonalbandpattern. Our results
indicated that in MDS the monoclonal appearance of PMN
PCR”’,?’
by
or by Southern
would not necessarily represent the total marrowreplacement by clonal progenitor cells.
We demonstrated that erythroid and nonerythroidprogenitors of MDS had a similar clonal constitution, which indicates that the partially committed or earlier stem cells capable of differentiating into at
least
both
erythroid and
myelomonocytic lineages were affected. In MDS, clonal
involvement of erythroid cells, whose clonality was not fully
studied because of the anucleated nature of erythrocytes, has
beenreported to be always accompanied by myeloidcell
i n ~ o l v e m e n t . ~ ~This
~ ~ “ ’was
~ ’ ’confirmed again in the present
study.
Many cytokines have been reported toimprove the cytopenias of MDS
Whether they can bring
about
qualitative improvement (polyclonal hematopoiesis) in MDS
is a matter of consideration. In the present study, we added
SCF to our culture system and analyzed the clonal origin of
resulting colonies. SCF, or designated as either mastcell
growth factor (MGF) or c-kitligand (KL), acts relatively
early in the scheme of normal
In addition,
it improves colony formation by MDS marrow cells in synergy with GM-CSF” or EPO,” although the responses vary
among patients.33In the present study, SCF enhanced colony
formation two to six times more in each patient as well as
normal individuals, but it did notalter the clonal constitution
of either erythroid or nonerythroid progenitor cells. Thus,
the additional colonies stimulated by SCF were not the result
of the preferential stimuli to nonclonal progenitor cells. It
is difficult to expect SCF will restore normal (polyclonal)
hematopoiesis in MDS patients. However, it cannot be ex-
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IN MDS
EMATOPOIETIC NONCLONAL
PMN
T cell
PMN
”
PMN
593
MNC
”
T cell
PMN
MNC
PMN
T cell
“
UPN
Fig 5. Clonal patterns of peripheral blood cells. DNA from PMNand either T cell or MNC wereamplified by nested-PCR. DNA in HI+) lanes
were precut with Hpall.
cluded that other cytokines, or their combinations, preferentially act on residualnonclonal progenitor cells, resulting
in qualitative improvement in MDS hematopoiesis. Present
clonal analysis is useful in evaluating the cytokine effects
in vivo as well as in vitro.
Whenever X-inactivation is used in clonal analysis, extreme ly~nization’~.”
should be considered. To rule out such
lyonization, we analyzed clonality of either T lymphocytes
or MNC from peripheral blood and compared the clonality
with that of PMN. Among three patients whose PMN showed
a monoclonal band pattern, two (UPN 1 and 3) had T lymphocytes of oligoclonal band patterns. MNC of one patient
with oligoclonal PMN (UPN 2) yielded a polyclonal band
pattern. In these three cases, acquired clonal evolution may
have occurred in marrow. In UPN 4,because her T lymphocytes showed a monoclonal band pattern identical to PMN,
we could not rule out completely the possibility that clonal
UPN 2
UPN 1
1
I.
11.7 kb
hematopoiesis resulted from extreme lyonization. However,
buccal mucosa of UPN 4 were composed of polyclonal cell
populations (data not shown), which might indicate pluripotent hematopoietic stem cell involvement in the patient. Circulating PMN and MNC of UPN S showed a similar polyclonal band pattern. Therefore, we could not ascertain that
acquired clonal evolution had caused the skewed clonal constitution of marrow progenitor cells.
As monoclonal hematopoiesis has been considered one of
the characteristics in MDS, the polyclonal appearance in
UPN S may seem extraordinary. However, this case may not
be so exceptional because polyclonal blood-cell populations,
other than lymphocytes, have been reported in some MDS
patients.I4.” Here, we demonstrated that polyclonal hematopoiesis can persist in MDS marrow. In addition, the clonal
composition of hematopoietic progenitor cells may be different from patient to patient. Thus, the heterogeneity of MDS
might be true also in the hematopoietic progenitor cell clonality.
Finally, it is of great interest whether the mosaicism of clonal
andpolyclonalprogenitorcellsisstableornotwithdisease
progression and whether remaining polyclonal progenitors affect the outcome with chemotherapy and cytokine stimulation.
Studies in this area are in progress in our laboratory.
REFERENCES
1.3kb
Fig 6. Clonal analysis of PMN by Southern blotting. DNA in H[+)
lanes werecut withHpall. Asingle band in H[+) lane of UPN 1 damonstrates monoclonal PMN, while the band pattern in H[+)lane of UPN
2 demonstratesthe extremely skewed clonal constitution.
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From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
1994 84: 588-594
Evidence for nonclonal hematopoietic progenitor cell populations in
bone marrow of patients with myelodysplastic syndromes
H Asano, H Ohashi, M Ichihara, T Kinoshita, T Murate, M Kobayashi, H Saito and T Hotta
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