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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2001-12-0188.
NEOPLASIA
From the Departments of Stem Cell Biology, Hematology,
and Clinical Genetics, Lund University Hospital, Sweden; the Divisions
of Hematology, Departments of Medicine, Karolinska Hospital and
Huddinge University Hospital, Karolinska Institutet, Stockholm,
Sweden; the Department of Hematology, Aarhus University Hospital,
Denmark; and the Department of Hematology, Rigshospitalet, Copenhagen,
Denmark.
Clonality studies of mature cells suggest that the primary
transformation event in myelodysplastic syndrome (MDS) most frequently occurs in a myeloid-restricted progenitor, a hypothesis supported by
recent studies of purified CD34+Thy1+
hematopoietic stem cells (HSCs) in cases with trisomy 8 (+8). In
contrast, we recently demonstrated that a lymphomyeloid HSC is the
target for transformation in MDS cases with del(5q), potentially reflecting heterogeneity within MDS. However, since +8 is known to
frequently be a late event in the MDS transformation process, it
remained a possibility that
CD34+CD38 Myelodysplastic syndromes (MDSs) are a
heterogeneous group of clonal disorders, characterized by ineffective
hematopoiesis and frequent progression to acute myeloid leukemia
(AML).1 The dysplastic changes and the resulting
cytopenias involve all mature myeloid but not lymphoid blood lineages,
and at variance with the hematopoietic stem cell (HSC) disease chronic
myelogenous leukemia,2 transformation to acute
lymphoblastic leukemia is extremely rare, suggesting that the primary
transformation event in MDS in most cases might occur at a multipotent
but myeloid-restricted progenitor cell level.3 This notion
has been supported by most clonality studies, since mature myeloid
cells are invariably part of the MDS clone, whereas B and T cells only
rarely are derived from the MDS clone.4-14 However, the
scarcity of evidence for mature lymphocytes being derived from the MDS
clone does not exclude the possibility that the primary transformation
event in MDS can occur at the HSC level. For example, the
transformation events in MDS may be incompatible with further lymphoid
differentiation and if so, the lymphocytes in patients with MDS may be
derived from residual normal HSCs or long-lived lymphoid progenitor
cells.14,15
Recently, major technological developments have allowed for prospective
identification and purification of human HSCs and progenitor cells
based on fluorescence activated cell sorting (FACS).16-18
In addition, their functional characterization has become possible by
improved in vitro and in vivo surrogate human HSC
assays.19-23 This progress will facilitate a new line of
studies in which the HSC and progenitor cell pools of patients with
various hematologic malignancies can be prospectively identified and
purified for further cytogenetic and functional studies. Unfortunately, the low frequency of phenotypically defined HSCs in healthy subjects as
well as in patients with MDS makes it a challenging task to obtain
sufficient cell numbers for functional studies. However, in a few
studies it has been possible to perform fluorescence in situ
hybridization (FISH) on FACS-purified HSC and progenitor cell
populations from patients with MDS to address whether these carry known
cytogenetic aberrations. Using this approach, we recently demonstrated
that a multipotent (lymphomyeloid) HSC is the likely primary target for
transformation in most MDS cases with 5q deletions.12 This
was based on the observation that more than 95% of the cells in the
CD34+CD38 Saitoh et al24 investigated MDS cases with trisomy 8, and
demonstrated in striking contrast to what we observed in cases with
del(5q) that whereas myeloid committed progenitor cells were invariably
part of the +8 clone, there was no evidence for involvement of the
phenotypically (CD34+Thy-1+) defined HSC pool.
Importantly, this lead to the conclusion that in MDS cases with trisomy
8 the transformation may predominantly originate in a committed myeloid
progenitor, and as a consequence it was proposed that this made
autologous HSC transplantation a meaningful approach in this patient
group.13,24
Based on these and other studies it is now commonly viewed that
MDS, being a heterogeneous disease, most often originates in a
multipotent but myeloid-restricted progenitor cell population, although
the primary transformation in some patients may occur at the
lymphomyeloid HSC level. Hence, the potential involvement of HSCs in
MDS remains a matter of controversy.3,14,15 However, the
absence of trisomy 8 in the CD34+Thy1+ HSC pool
of trisomy 8 patients does not exclude that this phenotyically defined
HSC population may be part of the MDS disease because the possibility
exists that the +8 aberration could be a secondary or late event in the
MDS transformation as previously proposed,25 potentially
occurring outside of the HSC pool. A functional evaluation of the HSC
population in trisomy 8 patients would have contributed to a better
understanding of whether the phenotypically defined HSCs were part of
the disease in these patients. However, such studies were not performed
by Saitoh et al,24 probably due to the limited number of
cells obtained. In fact, sufficient CD34+Thy-1+
cells for FISH analyses were only obtained in 3 patients. In the
present study, we have investigated the clonal involvement and
functional activity of the CD34+CD38 Patients
Purification of BM populations
FISH of FACS-purified BM populations
FISH on MGG-stained cells BM smears were stained with MGG and individual cells were morphologically identified (erythroid = normoblasts, granulocytic = promyelocytes + myelocytes + metamyelocytes + bands + segmented + eosinophils) and by unique coordinates they were reidentified by dual FISH analysis for del(5q) and +8 simultaneously, as previously described.28 When performing dual FISH on MGG-stained BM smears, the cut-off value for significant +8 findings was 2% to 3%.Hematopoietic growth factors Recombinant human (rh) megakaryocyte growth and development factor (MGDF), rh granulocyte-colony-stimulating factor (G-CSF), rh stem cell factor (SCF), rh interleukin 3 (IL-3), and rh granulocyte macrophage (GM)-CSF were generously provided by Amgen (Thousand Oaks, CA). Rh erythropoietin (Epo) was supplied by Boehringer Mannheim (Mannheim, Germany), and rh flt3 ligand (FL) was supplied by Immunex (Seattle, WA).Single-cell clonogenic assay As described previously,12,29 CD34+CD38+ and CD34+CD38 cells were seeded in Terasaki
plates (Nunc, Kamstrup, Denmark) at a density of one cell per well in
20 uL serum-depleted medium (X-vivo 15; BioWhittaker) supplemented with
1% bovine serum albumin (Stem Cell Technologies, Vancouver, Canada),
104 M 2-mercaptoethanol, and cytokines. Wells were scored
for cell growth after 11 to 13 days of incubation, as previously
described.27 A total of 120 wells were seeded per group,
but since the statistical chance (based on Poisson probability
distribution) of a well not receiving a cell is 37% by this method,
the maximum expected clones was 76.
Long-term culture-initiating cell (LTC-IC) assay Long-term cultures were either established with normal allogeneic BM MNCs according to previously described methods12,27 or with murine stromal feeders engineered to produce human growth factors (M2-10B4 and SI/SI mixed 1:1; kindly provided by D. E. Hogge, Vancouver, Canada).30,31 The murine stroma was established in 96-well collagen-coated microtiter plates with 5000 cells/well of each cell line, after irradiation with 8000 cGy and cultured in long-term culture medium (MyeloCult H5100; Stem Cell Technologies) with hydrocortisone 21-hemi-succinate 106M. CD34+CD38+ (250-25 000)
and CD34+CD38 (75-11 000) cells from
patients with MDS and healthy adults were added to the stroma layers
and cocultures were maintained at 37°C in high humidity and with 50%
medium exchange every week. After 6 weeks, nonadherent and adherent
cells were plated in methylcellulose cultures, supplemented with SCF,
GM-CSF, G-CSF, FL (all at 10 ng/mL), IL-3 (5 ng/mL), and Epo (5 U/mL).
Colony-forming cells (CFCs; readout of LTC-IC assay) were scored after
an additional 10 to 14 days in culture. Individual colonies were picked
and transferred to slides for subsequent FISH analysis.
Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) repopulating assay NOD/LtSz-SCID or NOD/LtSz-SCID 2-microglobulin-deficient (B2mnull) mice
(kindly provided by Dr L. D. Shultz, The Jackson Laboratory, Bar
Harbor, ME) were bred and housed under sterile conditions in
microisolator cages and given autoclaved food and acidified drinking
water (and for the NOD/LtSz-SCID B2mnull mice also
prophylactic trimethoprim-sulfadoxine [80/400 µg/mL]), 3 times a
week during both breeding and experiments. Mice were irradiated with
350 to 375 cGy from a 137cesium source at 8 to 12 weeks of age and thereafter given prophylactic ciprofloxacin (100 µg/mL) in the drinking water until analysis (6-8 weeks after
transplantation). Tail-vein transplantation/injection of hematopoietic
cells suspended in 0.5 to 1.0 mL of medium was performed within 4 hours
of irradiation. When less than 1 × 105 cells were
transplanted, 1 × 106 irradiated (1500 rad) accessory
cells (human MNCs or CD34-depleted cells) were coinjected. Femora and
tibiae were collected after asphyxiation with CO2, and
engraftment was investigated as described previously.12,31
Briefly, cells were stained with anti-human CD45-FITC and CD71-FITC
antibodies as well as anti-mouse CD45.1 (Ly 5.1)-PE. Mice that did not
receive transplants (negative controls) and mixtures of 0.5%
human cells in murine BM (positive controls) were always included. If
engraftment was detected by CD45/71 analysis (detection level < 0.05%), staining with anti-CD34-FITC, CD19-PE, CD33-PE/CD66b-FITC,
and CD15-PE was performed, including antihuman CD45-APC. 7-amino
actinomycin D (7-AAD; Sigma) was always included to exclude dead cells.
A minimum of 50 000 BM cells was examined for each sample. Only mice
positive for both myeloid and lymphoid human engraftment (defined
as > 10 positive events each per 50 000 viable BM cells) were
evaluated as positive. Gates were set so that samples incubated with
irrelevant isotype-matched control antibodies had a maximum of one
positive event per 50 000 BM cells analyzed. If no engraftment was
detected by flow cytometry or if the myeloid engraftment was low as
defined by flow cytometric analysis, BM cells (5 × 104
to 1 × 105) from NOD-SCID mice were plated in
methylcellulose with human-specific cytokines (50 ng/mL GM-CSF, 25 ng/mL IL-3, and 25 ng/mL SCF) and 5 U/mL Epo, resulting in no colony
formation of BM from NOD-SCID mice that did not receive
transplants (L.N. and S.E.W.J., unpublished data, April
1999). Granulocyte macrophage-colony forming units (CFU-GMs)
and erythroid-burst-forming units (BFU-Es) were scored after 10 to 14 days and transferred to slides for FISH analysis.
Involvement of the
CD34+CD38 cells, although representing less
than 0.1% of total BM cells.16,21,22 The
CD34+CD38 fraction also appears to harbor the
HSCs in patients with AML.32 Here we investigated the
coexpression of CD34 and CD38 in 10 MDS cases with +8 (Table
2). As expected, the mean frequency of
CD34+ cells in BM MNCs was slightly higher than in healthy
subjects, but as previously demonstrated for healthy
individuals,16 the majority of the CD34+ cells
in all investigated MDS cases with +8 coexpressed CD38 and only a
minority of the cells were found to be
CD34+CD38, in agreement with what we recently
observed in MDS cases with del(5q).12
CD34+CD38+ and
CD34+CD38 Whereas Thy-1 is known to be expressed on 5% to 25% of normal
CD34+ cells, and CD34+Thy-1+ cells
are highly enriched in HSC activity,17,33 its pattern of
expression in hematologic malignancies is less clear. In AML, Thy-1 is usually not expressed or only at very low levels on
CD34+ cells.34-36 Thus, it was of
interest to also investigate Thy-1 expression in patients with
MDS. In agreement with its usefulness as an HSC marker, the vast
majority of CD34+CD38 Functional evaluation of the HSC compartment in patients with MDS trisomy 8 The lack of CD34+Thy1+ cells carrying +8 in the study by Saitoh et al24 suggested that the HSC compartment in these patients with MDS may be normal. Although our FISH data indicated that a variable part of the CD34+CD38 HSC pool belonged to the +8 clone,
there was always a sizeable fraction that did not carry this
aberration. Therefore, as in the study by Saitoh et al,24
these may potentially represent a residual and sizeable normal HSC
pool, thereby providing a potential strategy for utilizing high-dose
chemotherapy followed by autologous transplantation in these
patients.13,24 However, the FISH data did not exclude the
possibility that CD34+CD38 cells disomic for
chromosome 8 are still part of the MDS clone (ie, the cells with
trisomy 8 may represent a subclone). Although phenotypic (and gene
expression) profiling is important for identifying HSCs, the presence
of normal HSCs can only be demonstrated through their unique functional
capacities.20 Normal HSCs show a characteristic response
pattern to nonredundant HSC active cytokines,29,37 and
reconstitute long-term cultures in vitro and immune-deficient mice in
vivo.19,38,39 Thus, we here functionally characterized the
CD34+CD38 (as well as the
CD34+CD38+) HSC compartment of MDS cases with
trisomy 8.
First, we tested the responsiveness of single
CD34+CD38
As previously demonstrated,19 normal
CD34+CD38
Of the trisomy 8 cases that demonstrated a complete lack of
LTC-IC activity, 3 were also investigated for potential in vivo HSC
activity in the NOD-SCID transplantation assay. Whereas 6 of 17 mice
that received transplants with normal BM CD34+
(168 000-500 000) or CD34+CD38
MDS cases with trisomy 8 have an intrinsic HSC deficiency, also involving HSC disomic for chromosome 8 The functional studies described above clearly suggested that in all trisomy 8 cases investigated, the large numbers of CD34+CD38 cells with disomy 8 were also
functionally deficient. To obtain more direct evidence for this notion
and to address whether the deficiency was intrinsic to the HSCs, we
next performed 2 lines of experiments. In the first, we addressed
whether the presence of MDS cells might negatively affect potentially
coexisting normal HSCs. Toward this aim,
CD34+CD38 cells were isolated from 2 patients
with MDS in whom the CD34+CD38 cells had no
LTC-IC activity (Table 4). These
CD34+CD38 cells were next mixed with
CD34+CD38 cells isolated from a healthy donor
and cultured in the long-term culture assay. In both cases (one
trisomy 8 and one del[20q]), the presence of MDS
CD34+CD38 cells, even at 10- to 100-fold
higher numbers than normal CD34+CD38 cells,
had no effect on the activity of LTC-IC derived from the normal cells
(Table 4). As expected, FISH analysis demonstrated that the LTC-CFCs
were without aberrations and derived from the healthy donor (opposite
sex) (L.N. and S.E.W.J., unpublished data, November 2001).
These data suggested that in the investigated trisomy 8 cases the vast
majority of CD34+CD38
There were 3 additional patients (no. 3, no. 5, and no. 13) who were
investigated by dual FISH combined with MGG staining (Figure
5). Also in all of these patients, all
cells either carried only del(5q) or both del(5q) and +8, whereas no
cells with +8 alone were observed. Thus, trisomy 8 appears to be a
secondary or late event in MDS, and although this might occur outside
of the HSC pool, the primary transformation appears to occur at the CD34+CD38
Allogeneic bone marrow transplantation represents the only
curative treatment for MDS but is rarely a realistic therapeutic option, due to the advanced age of most patients and the limited availability of donors.1,3 From the search for new
therapeutic modalities, autologous transplantation has emerged as a new
and potentially useful approach for treating MDS.3,40
Thus, the potential involvement and status of the HSC pool in patients
with MDS could be of considerable importance for the clinical outcome of autologous transplantation. As such, autologous HSC transplantation has been implicated as a potentially useful approach in patients with
MDS with trisomy 8, because the CD34+Thy1+ HSC
pool in these patients was found not to carry the +8
aberration,24 therefore potentially representing residual
normal HSCs. At variance with this, we had previously shown that the
CD34+CD38 Whereas none of the 12 previously investigated MDS cases with del(5q)
had more than 6% of cells in the CD34+CD38 Interestingly, in MDS cases with trisomy 8 at earlier stages of
disease, the CD34+CD38 As previously demonstrated for MDS cases with del(5q),
CD34+CD38 There are 2 lines of evidence that strongly support that the absence of
detectable normal HSC activity in CD34+CD38
We thank Drs Janet Nichol and Graham Molineux (both at Amgen) and Stewart Lyman (at Immunex) for generous contributions of cytokines for these studies, and Drs. P. D. Jensen (University of Aarhus), P. G. Nilsson (Department of Hematology in Lund), and I. Turesson (Department of Hematology in Malmö) for providing crucial BM samples. The assistance of Anna Fossum, Zhi Ma, and Gunilla Gärdebring in cell enrichment and sorting, Lilian Wittman in mouse work (all at the Department of Stem Cell Biology in Lund) and Bodil Strömbeck (Department of Clinical Genetics in Lund) for some FISH analyses, are highly appreciated. We also thank all patients and bone marrow donors for their contributions; the staff at the Department of Hematology for assistance; and Inge Olsson, Jan Westin, Bengt Sallerfors, Tor Olofsson, Theo de Witte, and Stefan Karlsson for helpful discussions and critical review of the manuscript.
Submitted December 6, 2001; accepted February 25, 2002.
Prepublished online as Blood First Edition Paper, April 30, 2002; DOI 10.1182/blood-2001-12-0188.
Supported by grants from the Swedish Cancer Society, the Cancer Society in Stockholm, The Crafoord Foundation, Georg Danielsson Foundation, Nilsson's Cancer Foundation, Åke Wiberg Foundation, John and Augusta Persson Foundation, John Persson Foundation, Tobias Foundation, Government Public Health (ALF) Grants, Skåne Landsting, and the Medical Faculty, University of Lund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Sten Eirik W. Jacobsen, Department of Stem Cell Biology, Biomedical Center, Klinikgatan 26, BMC B12, SE-221 85 Lund, Sweden; e-mail: sten.jacobsen{at}stemcell.lu.se.
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Thy-1+ hematopoietic
stem cell pool in myelodysplastic syndromes with trisomy 8
Top
Abstract
Introduction
20%
blasts). However, in these 2 patients the LTC-CFC activity was derived
from the +8 clone (L.N. and S.E.W.J., unpublished data, November
2001), and thus no evidence for normal HSC activity was
observed in the CD34+CD38
