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Biology of stem cells and myeloid progenitor
cells in myelodysplastic syndromes cells in myelodysplastic syndromes
Biology of stem cells and myeloid progenitor
© L.F.R. Span, Maastricht 2007
ISBN 10: 90-5278-600-3
ISBN 13: 978-90-5278-600-1
Layout: Tiny Wouters, Maastricht
Cover: Georges Span, Maastricht
Production: Datawyse | Universitaire Pers Maastricht
The studies described in this thesis were supported financially by grants from
Sacha Swarttouw Hijmans Foundation and NWO/FMW.
The author is grateful for the financial support provided by Amgen BV, Bristol-
Myers Squibb BV, Cephalon BV, Fresenius Kabi Nederland BV, Janssen-Cilag
BV, Mundipharma Pharmaceuticals BV, Novartis Oncology, Ortho Biotech, Pfizer
BV, Schering Nederland BV, Schering-Plough BV.
B Bi io ol lo og gy y o of f s st te em m c ce el ll ls s a an nd d m my ye el lo oi id d p pr ro og ge en ni it to or r
c ce el ll ls s i in n m my ye el lo od dy ys sp pl la as st ti ic c s sy yn nd dr ro om me es s
Een wetenschappelijke proeve op het gebied van de
ter verkrijging van de graad van doctor
aan de Radboud Universiteit Nijmegen
op gezag van de Rector Magnificus prof. dr. C.W.P.M. Blom,
volgens het besluit van het College van Decanen
in het openbaar te verdedigen op woensdag 7 februari 2007
om 13.30 uur precies
Lambert François Rudolphe Span
geboren op 3 april 1963
Prof. dr. T.J.M. de Witte
Dr. R.A.P. Raymakers
Prof. dr. A.H.M. Geurts van Kessel
Prof. dr. E. Vellinga
Prof. dr. J.H.J.M. van Krieken
Voor Swanny, Florence, Thomas en mijn ouders
Ter nagedachtenis aan Adriana Wilhelmina (“Mientje”) Liebregts
Chapter 1 Biology of stem and myeloid progenitor cells in
Cancer Treatment and Research. 2001;108:45-63
Chapter 2 Apparent expansion of CD 34+ cells during the evolution of
myelodysplastic syndromes to acute myeloid leukemia
Chapter 3 The dynamic process of apoptosis analyzed by flow
cytometry using Annexin-V/PI and a modified in situ end
Chapter 4 Bone marrow mononuclear cells of MDS patients are
characterized in vitro by hyperproliferation and increased
apoptosis independently of stromal interactions
Chapter 5 Programmed Cell Death is an intrinsic feature of MDS
progenitors, predominantly found in the cluster-forming cells
Experimental Hematology 2005;33:435-442
Chapter 6 Caspase-inhibitors decrease Programmed Cell Death of
CD34+ cells from MDS patients without restoration of a
normal in vitro growth pattern
Chapter 7 Summary
Leukemia Research, Submitted
apoptosis or programmed cell death
abnormally lokalised immature precursors
acute myeloid leukemia
alkaline phosphatase antialkaline phosphatase complexes
Bcl-xL/Bcl-2 associated death promoter
Bcl-2 associated X gene
B-cell lymphoma/leukemia-2 gene
a Bcl-x isoform that inhibits PCD
bone marrow mononuclear cells
bovine serum albumin
Cluster of Differentiation
colony-forming unit – granulocyte, erythrocyte, monocyte,
colony-forming unit – granulocyte, macrophage
this proto-oncogene encodes a transcription factor (Myc) that
promotes growth, proliferation and apoptosis
cytotoxic T lymphocyte
double distilled water
Early Growth Response gene-1
Fas associated death domain (= MORT-1)
Fas associated phosphatase-1
CD95 or APO-1
Fas receptor or Fas
Fc fragment of immunoglobulin
modified ISEL technique for FCM
fetal calf serum
(fluorescence) in situ hybridization
temporarily non-proliferating cells within G0 of cell cyclus
the interval between mitosis (cell division) and S-phase
(DNA replication) of the cell cycle
granulocyte colony-stimulating factor
granulocyte-macrophage colony-stimulating factor
hematopoietic stem cell(s)
human stem cell factor
IL-1? converting enzyme
Dulbecco's edium, Iscove Modification M 's
International Prognostic Scoring System
interferon regulatory factor-1
in situ end labeling
in situ hybridization
labeling index or percentage cells in S-phase
long-term bone marrow cultures
Multidrug Resistance gene 1
normal bone marrow
RA / RARS
number (or n), or normality (for solutions)
Natural Killer cells
total number of aggregates
number of aggregates at day x
peripheral blood stem cell or phosphate buffer solution
peripheral blood stem cell transplantation
rogrammed ell eath PCD
refractory anemia / RA with ringsideroblasts
refractory anemia with excess of blasts (in transformation)
revolutions per minute
RPMI medium was developed by Moore et. al. at Roswell
Park Memorial Institute
secondary AML after MDS
stem cell factor
single-cell single-well (assay)
standard error of mean
soluble Fas Ligand
soluble Fas or FasR
tris buffer solution
tris buffer solution with Tween 20
cell cycling time
cell doubling time
tumor necrosis factor-?
tumor necrosis factor receptor 1
duration of S-phase or DNA synthesis time
terminal deoxynucleotidyl transferase nick-end labeling
very late antigen
World Health Organization
X chromosome inactivation pattern(s)
a cell-permeable, irreversible inhibitor of caspase-1, -3, -4,
a cell-permeable, irreversible inhibitor of caspase-3, -6, -7,
-8, and -10.
Biology of stem and myeloid progenitor
cells in myelodyplastic syndromes cells in myelodyplastic syndromes
Biology of stem and myeloid progenitor
LFR Span, TJM de Witte
Cancer Treatment and Research. 2001;108:45-63
General introduction ?15
The myelodysplastic syndromes (MDS) are hyperproliferative, acquired clonal
stem cell disorders, associated with massive intramedullary apoptosis or
programmed cell death (PCD). A leukemic phenotype, mainly characterized by
an increase of blasts showing differentiation arrest, is gradually observed, as
MDS progresses from low-risk (LR-) to high-risk MDS (HR-MDS). Three
interacting compartments can be distinguished in MDS bone marrow (BM); the
polyclonal, residual normal hematopoiesis, the monoclonal preleukemic
compartment, and the blastic leukemic compartment. Within LR-MDS,
monoclonal hematopoiesis dominates leukemic blast cell proliferation (<5%),
whereas this pattern is reversing during MDS evolution to acute myeloid
leukemia (AML). This shift in the balance of proliferation versus apoptosis has
to be applied constantly to these three interacting compartments in MDS. For
illustration, the following example and nomenclature is used. Stem Cell Factor
(SCF) is a major factor to induce differentiation and to mediate the transition
from the earliest CD34 negative (CD34?) stem cells to the more differentiated
CD34 positive (CD34+) stem cells, whereas IL-6 promotes proliferation and
maintains self-renewal of CD34+ stem cells. The balance and interaction
between these cytokines may play different roles in normal, monoclonal, and
leukemic hematopoiesis ("static profile" of these compartments). Furthermore,
these cytokine levels, their receptor density, and/or their receptor-ligand
interaction may change within each hematopoietic compartment as MDS
progresses, also changing the balance and interaction of these cytokines
between these three pools ("dynamic profile"). Genetic or phenotypic changes
of the malignant pool and changing interactions with the environment (stroma
or different accessory cells) also cause a complex dynamic change in different
interactions (cell-cell, cell-stroma, stroma-stroma) between these three pools
as MDS progresses ("complex-dynamic profile").
This review describes these complex-dynamic profiles of stem cells and
progenitors in MDS. Emphasis is put on the origin of monoclonality and its
implications in MDS (Part I) and phenotypic-functional studies to understand
the biology (balance of proliferation and apoptosis) of MDS in evolution
(Part II). Part III of this chapter discusses the FasR/FasL system as one of the
most important members of the nerve-growth factor receptor family for carrying
apoptosis signal transduction.
Part I. Clonality in MDS
Dysplastic features and an increase of blasts found in hypercellular BM
characterize MDS. The International Prognostic Scoring System (IPSS)1, based
upon the percentage of marrow myeloblasts, cytogenetic characteristics, and
the number of cytopenias, defines prognosis and survival of MDS patients with
more accuracy than the original French-American-British (FAB) classification.
MDS progression to AML is determined by further accumulation of genetic
defects in the myelodysplastic clone. Early studies suggested that these
aberrant clones may originate in a more committed myeloid stem cell (CFU-
GEMM: colony-forming unit–granulocyte, erythrocyte, monocyte) in most
patients, evaluated by immunophenotyping and (fluorescence) in situ
hybridization or (F)ISH2-4. More recent studies proved that cytogenetically
aberrant cells could also be detected in the primitive stem cell pool5,6. Mehrotra
et al.5 found cytogenetically aberrant cells in a primitive (CD34+lin?) stem cell
compartment. The percentage abnormal cells was not associated with
compartment expansion, indicating that these aberrant primitive hematopoietic
cells do not show a leukemic phenotype (growth and survival advantage). This
primitive compartment with high Multidrug Resistance gen 1 (MDR1)
expression accounts for the high relapse rate of MDS patients treated with
intensive chemotherapy and autologous BM transplantation.
In general, it is hypothesized that a first hit causes inactivation or deletion of
tumor suppressor genes (e.g. Interferon regulatory factor 1 (IRF-1) and Early
Growth Response 1 (EGR-1) at 5q31-33 region), DNA repair genes (at 7q22
region) or, although less frequently, activating mutations in a proto-oncogene
(e.g. N-ras and its association with chromosome 7 deletions). This first hit will
subsequently lead to a "controlled" growth advantage of this "damaged" stem
cell and its progeny over the normal pool of stem cells7. MDS in preleukemic
phase detects monoclonality (in females) by X-chromosome inactivation
studies (see below), which may occur before the development of karyotypical
abnormalities. The growth advantage of these preleukemic CD34+ cells and its
progeny may be caused by an increase of the number of S-phase cells with or
without a substantial survival benefit8. This genetically altered progenitor cell
pool is more prone to additional mutations or deletions ("genomic instability").
S-phase cells are more susceptible to detrimental DNA events because of their
status of unpacked and uncoiled DNA and intense DNA synthesis with less
DNA repair time. The final behavior of the leukemic clone in MDS is determined
by its overall make-up of activated genes. Patients with balanced chromosomal
translocations seem more likely to present with overt leukemia than patients
with unbalanced chromosomal abnormalities9,10.
Interestingly, patients with and without abnormal karyotype have no different
levels of overall apoptosis11. It is therefore tempting to speculate that
monoclonality by itself induces immune responses leading to overt apoptosis.
This apoptotic process is also conferred to the normal, polyclonal
hematopoiesis and stromal tissues as innocent bystanders (see Part II). As
polyclonal blood cells are dying intramedullary, relatively more apoptosis-
General introduction ?17
resistant blood cells with normal or dysplastic appearance in the peripheral
blood are found as the progeny of monoclonal hematopoiesis12. Furthermore,
the percentage cytogenetically aberrant BM blasts is always considerably
higher than the BM mature granulocytes indicating a partial maturation arrest
(and decreased PCD) of monoclonal hematopoiesis in MDS13. Above all, anti-
apoptotic therapy in MDS patients sometimes results in disappearance of
cytogenetically aberrant clones and resumption of polyclonal hematopoiesis14.
The role of FasR/FasL in MDS is probably a double-edged sword: a tool of
immune surveillance by NK (Natural Killer) cells and/or cytotoxic T cells with
enhanced membrane-bound FasL (mFasL) attacking the preleukemic clone(s),
which in their turn show decreasing FasR and increasing FasL expression
during leukemic progression. This process gradually leads to an escape of the
leukemic cells (with high FasL expression) from immunoregulatory cells and
probably contributes to progressive PCD of normal and monoclonal
preleukemic hematopoiesis with enhanced FasR expression (see part III).
The HUMARA assay which uses a polymorphic gene on the X-chromosome
showing a high rate of heterozygosity (>90%) is the most used assay to
study X Chromosome Inactivation Patterns (XCIP) to detect monoclonality. The
presence of monoclonality is an early feature in MDS. However, differentiation
from constitutional excessive Lyonization and acquired skewing associated
with increasing age, is a major limitation in interpretation of these assays (total
skewing 15-40%). The use of T lymphocytes as control cells and sequential
analyses may solve this practical problem. On the other hand, no specific
genetic marker is needed for assessment of monoclonality with XCIP. The
different XCIP assays used on different sorted subsets of BM and blood in
MDS patients have shown monoclonality originating in a primitive
(CD34+Lin?/Thy1+) or early committed (CD34+CD33+) stem cell.
Part II. Biological features of CD34+ cells and their myeloid
progeny in MDS in evolution
Characteristics of CD34+ cells in MDS
The percentage CD34, CD33, and CD13 positive bone marrow mononuclear
cells (BMMNC) increased as patients progressed to HR-MDS, and correlated
with shorter survival15-18. Also co-expression of CD13 (mean ?90%) was
significantly increased in MDS CD34+ cells. This was associated with a
predominant outgrowth of colony-forming units–granulocyte, macrophage
(CFU-GM), usually showing undifferentiated clusters, as hardly any erythroid
aggregates were found19. Furthermore, abnormally high ratios of pro- versus
anti-apoptotic proteins (c-Myc/Bcl-2 and Bax+Bad/Bcl-2+Bcl-x) were found
within the CD34+ cells of especially LR-MDS patients20,21. These ratios
reversed in advanced MDS and AML.
The size of the CD34+ pool is rapidly increasing during MDS progression in the
majority of patients8, and a concomitant rise in the number of aberrant blasts
occurred, as was detected by flow cytometry (FCM) side scatter and CD45
expression18. The morphologically "normal" CD34+ blast cells may contain
monoclonal, cytogenetically normal CD34 cells, but this remains to be proven.
The presence of circulating CD34+ cells in MDS correlated with leukemic
progression, even better than cytogenetics and CFU-GM growth in vitro
may indicate that cell-stroma interactions have changed in HR-MDS, and this
may contribute to leukemic evolution.
Proliferation of CD34+ cells and their progeny in MDS
In vivo thymidine analogue (BrdU/IUdR) incorporation studies in MDS
patients23,24 followed by BM immunohistochemistry (IH) have shown an
increment of overall proliferation of BMMNC. These studies demonstrated a
higher than normal overall myeloid growth fraction (GF: median percentage
S-phase cells 25-30%, range 13-49%) with a decreasing trend towards
HR-MDS. Furthermore, the total cell cycling times (Tc) increased when RA
progressed to RAEB-t (Tc of 37.5 and 56.6 hours, respectively). Within the
myeloid compartment, a rapid increment of the percentage of CD34+ cells (from
1.67 to 8.69% from LR- to HR-MDS, respectively) and the percentage CD34+
cells in S-phase (from 0.19 to 0.43%, respectively) was observed during the
evolution of MDS8. A concomitant rise was also found in the percentage
proliferating CD34+ cells within the proliferating myeloid compartment during
MDS progression (from 0.35 to 1.44%, respectively). These patterns clearly
illustrate a growth advantage within the CD34+ pool, which only partially
explains the exponential growth of the size of the CD34+ pool during MDS
evolution. Using FCM with Ki-67 (a proliferation marker), Parker et al.20 found
increasing percentages of proliferating (G1) CD34+ cells (range 10-70%) within
the (growing) CD34+ compartment during MDS progression, as the percentage
S-phase cells in the CD34+ pool was hardly changing in our study (mean
5-10%)8. As the nuclear antigen Ki-67 is not an excellent proliferation marker
for myeloid cells and it only distinguishes non-cycling (G0) from cycling (G1)
cells, the differences between these two studies can be explained by enhanced
PCD of CD34+ cells in G1. Thus, the CD34+ compartment expands as MDS
progresses with a tendency to cycle slower than their more mature CD34?
progeny. Furthermore, also the differentiation arrest in leukemic blasts18, and
their progressive survival benefit account for this expansion of CD34+ cells and
blasts. It is important to stress that proliferation and differentiation are
General introduction ?19
progressively uncoupled mechanisms during the evolution of the leukemic
clone(s) as MDS progresses.
Proliferation and apoptosis of CD34+ cells and their progeny in
MDS: dynamic profiles in vitro
In vitro studies with BMMNC of MDS patients have shown decreased colony
and increased cluster formation with slower growth kinetics, delayed and
disturbed differentiation or arrest at the stage of myelo-monocytic blasts25, as
well as increased apoptosis26. This leukemic growth pattern in vitro was
associated with an increase of blasts and CD34+ BM cells, and correlated with
a higher incidence of leukemic transformation with shorter survival15,27,28. We
studied the profiles of proliferation and PCD (by ISEL) of BMMNC of MDS
patients in vitro26 The proliferation, defined by the total number of clusters and
colonies, was initially enhanced as compared to normals. But this was rapidly
followed by a concomitant increased apoptosis: 75% of clusters and more than
40% of colonies showed more than 50% PCD. In contrast, normal controls
showed a median PCD of 50% in clusters and 17% in the colonies. AML
patients showed delayed and low colony growth in vitro, because of enhanced
apoptosis at cluster level (60-80%) compared to a relative low PCD in colonies
(20%). Interestingly, some colonies of AML patients showed no apoptosis at
Single cell assays of CD34+ BM cells in MDS showed a similar biological
profile: increased proliferation and apoptosis at cluster level and decreased
colony formation showing decreased cell numbers. These colonies showed
less overall PCD than normal colonies29. As neither stromal interactions nor
accessory cell influences are involved in this system, probably (pre)leukemic
clones with longer cell cycling time (43.7 hours in MDS versus 33.8 hours in
normal colonies) and less PCD can evolve in this system. It implies that the
patterns observed are intrinsic properties of MDS progenitor cells. (F)ISH
studies have to be performed to distinguish growth patterns of chromosomal
aberrant from normal clones.
Long-term bone marrow cultures (LTBMC), analyzing MDS stromal influences
on normal CD34+ cells, have shown defective surface coverage and support in
promoting proliferation and differentiation30, leading to increased levels of FasR
and apoptosis31. In contrast, Deeg et al.32 found stimulatory effects of MDS
stroma on normal CD34+ or MDS CD34+ cells. They suggested that accessory
mononuclear (non-stromal) cells or abnormal hematopoietic precursors in the
non-adherent marrow fraction provided the inhibitory effects as they produced
tumor necrosis factor-? (TNF?) at maximal levels.
LTBMC (with normal stroma) were capable of detecting latent subclones with
abnormal karyotypes in the majority of MDS patients with normal cytogenetics.
In some (25%) patients these karyotypes also became apparent in vivo.33.
Furthermore, LTBMC detected profound deficiencies in the number of
secondary colony-forming cells and in long-term proliferation of multipotent
MDS progenitors, together with disturbed differentiation and stromal
Stroma-free LTBMC with a combination of four growth factors (GFs) showed a
normal expansion of MDS progenitor cells with normal or dysplastic
differentiation in 50% of cases. Complete unresponsiveness and progressive
leukemic growth with 100% immature blasts was found in 30% and 20% of
MDS cases, respectively36. Furthermore, Novitzky et al.30 showed that the
subgroup of MDS patients with the highest overall BMMNC apoptosis had the
best clonogenic growth in vitro and showed the best response to anti-TNF?
therapy in vivo. This shows that the inhibitory and PCD-inducing cytokines and
their corresponding cells outweigh their stimulating counterparts in MDS
marrows. A high proliferative potential continues to be present in MDS
Several mechanisms may explain these altered growth patterns in vitro. MDS
CD34+ cells with enhanced co-expression of CD13 form predominantly
nonerythroid clusters with impaired differentiation19. MDS progenitors show a
diminished response to granulocyte colony-stimulating factor (G-CSF)25, and
granulocyte-macrophage colony-stimulating factor (GM-CSF)37, which could be
reversed by supersaturating doses in some patients38. Stem cell factor (SCF)
promotes cluster growth, whereas in combination with other GFs
undifferentiated colonies are promoted. SCF may in part be responsible for the
growth advantage of MDS clonogenic cells over normal blasts, although no
differences in c-kit expression were observed28,39,40. Enhanced apoptosis
detected in the progeny of BMMNC and CD34+ cells of MDS patients may
explain this decreased colony formation. In serum-free cultures and LTBMC,
MDS CD34+ cells showed poor or no growth of differentiated colonies,
irrespective of their growth type, suggesting the defective support of accessory
and stromal cells31,40. Influences of stromal and accessory cells are not the only
explanation of increased PCD as it was also found in single cell assays of MDS
CD34+ cells29. Differentiation between cytokine-mediated and/or FasR-FasL
mediated PCD is warranted!
Apoptosis of CD34+ cells and their progeny in MDS: dynamic
profiles and controversies
Apoptosis can be triggered by a variety of circumstances like growth factor
deprivation, receptor interaction like tumor necrosis factor receptor 1 (TNF-R1)
and FasR, and cell damage causing molecular and or genomic damage
beyond repair. Several quantitative techniques have been developed to
General introduction ?21
measure apoptosis like immunohistochemistry (IH) or flow cytometry (FCM) by
in situ end labeling (ISEL) or TdT-mediated dUTP nick-end labeling (TUNEL).
ISEL and TUNEL detect specific DNA fragmentation products developed after
the activation of different endonucleases. These techniques proved enhanced
intramedullary PCD41,42, as it was postulated before43.
Overall apoptosis detected by the ISEL technique on plastic embedded BM
biopsies was excessive in MDS. More than 50% of patients showed more than
75% ISEL+ cells41,44. In general, less apoptosis was found with TUNEL (mean
range 12-46%), especially when BM smears were used30,45-48. The detection of
PCD by morphology (±3%) underestimates the amount of apoptosis11. This
phenomenon is explained by the short duration of the apoptotic process. In
addition, early apoptotic cells present phosphatidylserine (PS) on their outer
membrane, signaling macrophages for engulfment, often before clear apoptotic
morphology can be detected. Apoptosis was observed in clusters of marrow
cells in BM biopsies and the amount of PCD was positively and significantly
correlated with the level and localization of TNF? expression23. Both trilineage
parenchymal and stromal cells are dying31,41. The number of macrophages was
clearly increased, showing massive ISEL-positive apoptotic bodies of captured,
dead cells23. PCD was inversely correlated with leukocyte count11.
Overall apoptosis of BMMNC measured by TUNEL on BM aspirates (range
20-46%) and BM biopsies (range 47-69%) is high in LR-MDS and decreases
during progression of MDS45,46,48,49. High rates of overall PCD were correlated
with low Bournemouth scores46 and were significantly correlated with low blast
numbers48. In contrast, a large number of BM biopsies treated with ISEL
showed massive apoptosis (>75%) in most HR-MDS patients. ISEL-positivity
(ISEL+) decreased towards intermediate levels (range 33-67%) in LR-MDS,
although both high and low ISEL positivity was found in LR-MDS23,41. The most
likely explanation for these discrepancies in PCD is the difference in used
material. More apoptotic cells in MDS marrow aspirates were found in the high-
density fraction of mononuclear cells than in the mostly used low-density
fraction50. A considerable amount of apoptotic cells is damaged and lost during
the work-up of marrow aspirates. Differences in the detection of different DNA
fragmentation products by ISEL and TUNEL is also a fair explanation42.
Another explanation may be the heterogeneity of MDS. The apoptotic degree is
different in the various compartments (CD34+ vs. CD34?, and leukemic vs.
monoclonal vs. polyclonal) in time. These compartments also change in size
during MDS progression.
In MDS, various levels of apoptosis within the CD34+ pool were found with
different techniques. Massive PCD of CD34+ progenitors was found in LR-
MDS, as it decreased towards HR-MDS20,21. Parker et al.20 showed excessive
apoptosis (median range 50-60%) by FCM using Annexin-V (AnV), whereas
Rajapaksa et al.21 showed a sub-G1 peak of 9%, both detected in the CD34+
subset in early MDS. In contrast, TUNEL performed on cytospin preparations of
sorted CD34+ cells of MDS patients showed a lower mean PCD of 24%, not
significantly different from normals. In general, higher values were found in LR-
versus HR-MDS30. Increased c-myc/Bcl-2 ratios of CD34+ cells were correlated
with enhanced PCD in LR-MDS patients21. In addition, pro-apoptotic (Bax+Bad)
versus anti-apoptotic (Bcl-2+Bcl-x) ratios were increased in CD34+ cells of
patients with LR-MDS. Disease progression was associated with significantly
reduced ratios, due to increased Bcl-2 and a reduction in Bad expression20.
Surprisingly, as these ratios play an eminent role as molecular death switches,
they were not associated with apoptosis measured by AnV, whereas they were
inversely correlated with IPSS score and cytogenetic risk group. AnV detects
PS on the outer membrane of cells. This is probably a marker before the point
of "no-return" of PCD, as a fraction of thawed AnV+CD34+ cells showed
proliferation in single cell assays51.
Conflicting results were found when CD34+ and CD34? cell populations were
compared with TUNEL by FCM and ISEL by IH52. In general, CD34? cells
showed more PCD with TUNEL than CD34+ cells, but this difference was not
significant, whereas 56% CD34? cells versus an occasional CD34+ cell showed
ISEL+ in BM biopsies. Different types of nuclear endonucleases found in CD34+
and CD34? cells causing different DNA fragmentation products may explain
this. Above all, ISEL positivity was decreased in blast clusters of advanced
MDS and in AML blasts in BM biopsies41.
The overall balance of these dynamic profiles between increased proliferation
and apoptosis results in ineffective hematopoiesis with cytopenias in the
peripheral blood. A significant positive correlation was observed between the
degree of PCD and proliferation41. Also anti-PCD treatment studies14 clearly
showed that PCD and proliferation were correlated phenomena. Parker et al.20
found the following apoptosis/proliferation ratios in the MDS CD34+ cells: 2.08
(RA/RARS), 1.14 (RAEB), and 1.7 (MDS-AML). Maximum PCD was found in
RA/RARS, whereas proliferation peaked in RAEB, and both processes
declined towards MDS-AML. “Signal antonymy” is an unique feature for MDS53.
It means that the cell is dying in S-phase as a result of concomitant
engagement into incompatible pathways like proliferation and cell cycle arrest.
Signal antonymy in MDS (mean of 54% of S-phase cells) was found in all
hematopoietic lineages as well as stromal cells. The explanation for this
phenomenon is still unknown.
Complex dynamic profiles in MDS: interactions
Growth factors and cytokines play an important role in the apoptotic processes
in MDS. In general, GFs can be considered as survival factors. The end result
is determined by the balance between levels of positive and negative
General introduction ?23
hematopoietic GFs and cytokines and their receptor status. Enhanced
expression of TNF? and IFN? was detected in BM biopsies of MDS
patients23,54. TNF? expression was significantly correlated with PCD. TNF?-
induced cytotoxicity is mediated by reactive oxygen intermediates generated in
the mitochondrial respiratory chain. Anti-apoptotic therapy by TNF?-lowering
regimens in MDS patients resulted in decreased TNF? levels followed by
decreased PCD in BM, and clinical responses in 40-50% of patients14,30,55.
These results led to Raza's postulation of a dual role of elevated TNF? (and
IL-1?) levels in the hematopoiesis of MDS patients: stimulation of proliferation
of CD34+ stem and early progenitor cells, but inducing apoptosis in their CD34?
progeny44. One contributing link interacting in this paradigm has been found:
TNF? (and IFN?) upregulates FasR/CD95 expression, which is one of the main
pathways of introducing cell death signals to cells. Sometimes a decrease or
disappearance of cytogenetically aberrant clone(s) was observed during TNF?-
lowering therapy14. These effects suggest that anti-TNF? treatment may favor
normal rather than aberrant hematopoiesis.
Part III. The Fas/FasL system in MDS
Introduction of normal physiology
All members of the nerve growth factor receptor family play dual roles as they
can trigger both apoptosis as well as proliferation56. One of the members is Fas
(CD95, APO-1), a 45kDa type I transmembrane glycoprotein. The Fas receptor
(FasR, or Fas) is normally expressed on a wide range of mature blood cells
(monocytes, neutrophils, NK cells, B and T lymphocytes), and highly expressed
on activated lymphocytes. In contrast, Fas is weakly expressed on immature
BM cells. CD34+/CD38+ cells have two-fold higher expression than
CD34+/CD38? cells57-59. The receptor density increases from early CD34+ stem
cells to more mature progenitors, and it is particularly upregulated on
proliferating myeloid progenitors60. Cytokines known to mediate proliferation,
maturation, and survival of hematopoiesis facilitate negative growth regulation
by the FasR pathway in activated cells60. This effect could serve as a negative
feedback mechanism by T cells on activated hematopoiesis61,62. These findings
suggest that the Fas/FasL system plays a role in the homeostasis of
hematopoiesis63. Furthermore, Fas expression is upregulated in a dose-
dependent fashion in IFN? and TNF? treated marrow CD34+ stem cells and it
facilitates FasR-induced PCD57,58. The combination of TNF? and IFN? had a
synergistic effect on the induction of Fas expression on progenitors57. Activated
peripheral blood mononuclear cells were able to produce soluble Fas
isoforms64. Soluble Fas (sFasR) inhibits apoptosis in vitro 65.
Fas Ligand (FasL) is a 40kDa type II transmembrane protein. FasL is
predominantly expressed in activated cytotoxic T cells (CTL's), B cells, and NK
cells, but it is also expressed on monocytes, neutrophils and tumor cells.
Membrane-bound FasL (mFasL) induces PCD by trimerization or cross-linking
of the Fas receptor in some Fas-expressing cell lines or memory T cells66. It
works as a cytotoxic effector molecule of CTL and NK cells, and probably of
AML tumor cells. Membrane-bound FasL can be cleaved into a soluble form
(sFasL) by a metalloproteinase67. Membrane-bound FasL is more potent in
promoting PCD than sFasL68. The shedding of FasL from the membrane is a
mechanism for downregulating its killing activity: sFasL competitively inhibits
the killing of T cells by mFasL68,69. Above all, Josefsen et al.59 found that sFasL
promoted cell survival of human BM CD34+CD38? progenitor cells by
suppressing PCD in suspension cultures as well as in single cell assay,
whereas PCD was slightly increased in the more mature CD34+CD38+ cells.
These studies demonstrate that the delicate balance between mFasL and
sFasL levels represents a (paracrine and/or autocrine) regulator of early
hematopoiesis: survival and proliferation promotion by sFasL versus apoptosis
induction by mFasL.
Interaction of Fas with its natural ligand (FasL) or with agonistic anti-Fas
monoclonal antibodies (like CH11) causes homotrimerization of CD95 and
triggers PCD by activation of the FADD/MORT-1 cascade70. Concurrent
expression of Fas and FasL on the same cell leads to PCD after interaction by
membrane folding, although monocyte-derived macrophages could escape
from spontaneous or anti-Fas IgM induced apoptosis71.
Enhanced Fas expression on BM progenitors seems to play a role in ineffective
hematopoiesis57. They showed that IFN? and TNF? mediated suppression of
colony formation from immature (CD34+CD38?) and mature (CD34+CD38+)
progenitors was enhanced by FasL without the presence of accessory cells.
IFN? and TNF? cause cell cycle inhibition of hematopoietic cells, upregulate
FasR expression on CD34+ cells, and induce ICE expression in these cells
which subsequently led to PCD in vitro when CH-11 was added57,58,72. Above
all, tumor cells with FasL expression escape from the T cell-mediated immune
surveillance, while they maintain the ability to induce Fas-mediated apoptosis
in normal cells, especially in activated lymphocytes73,74.
The Fas/FasL system in MDS
Immunohistochemical (IH) stained MDS BM sections showed positive staining
for Fas (and FasL), whereas BM samples of normals showed no staining47.
These findings were confirmed by RT-PCR for Fas (and FasL) mRNA18,47. Fas+
cells were found in all cell lineages, including CD34+ cells. Also Lepelley et al.45
observed increased Fas expression in BM cells (by IH) in about 40% of MDS,
General introduction ?25
whereas a variable proportion of blasts showed weak Fas expression. Gersuk
et al.18 found increased Fas expression of BMMNC by FCM in MDS. They
observed that considerably more CD34+ blasts showed Fas expression in MDS
as compared to normal BM (87% vs 25%), but Fas expression intensity on
CD34+ cells was negatively correlated to the BM blast number. Leukemic blasts
apparently loose Fas expression with progression of MDS46. Interestingly,
significantly more CD3+ activated T cells with Fas expression were found in
MDS BM in comparison to normal BM18.
Regarding the function of Fas, not all FasR+ BM cells showed TUNEL
positivity47. Furthermore, MDS BMMNC showed increased caspase-3
mRNA18,75 with a lower to absent FAP-1 expression, which is an inhibiting
modulator of the FasR signal transduction pathway76. Bouscary et al.48 found
clearly enhanced apoptosis by TUNEL associated with significantly increased
levels of caspase-3 activity and low blast numbers in LR-MDS patients.
Although overall Fas expression on hematopoietic progenitors was increased in
MDS, it was not correlated with FAB subtype, the Bournemouth score,
apoptosis rates or peripheral cytopenias45,46. In contrast, in vitro culture studies
in MDS have shown decreased clonogenic capacity of CFU-GM and the
involvement of enhanced Fas expression on proliferation and PCD18.45,46,77. The
erythroid lineage seems to be more sensitive for Fas-mediated apoptosis than
the myeloid lineage45,46,78. Also LTBM cultures with MDS stroma have shown
defective support in promoting proliferation and differentiation in combination
with increased levels of Fas and apoptosis of these normal progenitors31.
Higher levels of TNF? and sTNF-R1 were found in marrow plasma of MDS
patients as compared to normals18. The addition of anti-TNF? mAb or soluble
rhuTNFR:Fc to Dexter cultures increased colony numbers18.
FasL expression in MDS was increased in BM cells of all lineages, irrespective
their maturation state, but it was even higher in AML blasts47,78. This increase in
FasL expression was significantly correlated with FAB subtype, the number of
abnormal metaphases, and survival78. Furthermore, overall FasL expression in
de novo AML was comparable to AML after MDS78, whereas primary MDS had
significantly lower FasL+ aberrant blasts compared to secondary MDS18.
Gersuk et al.18 observed variable and increased amounts of FasL on MDS
CD34+ blasts in contrast to normal CD34+ cells. FasL expression was inversely
associated with TNF? levels and Fas expressing during MDS progression.
Furthermore, considerably more BM FasL+ CD3+ cells in MDS (17%) were
found as compared with normal BM (2%)18. The majority of apoptotic cells by
TUNEL were also FasL+ with the exception of macrophages47. Macrophages
showed considerably more staining for FasL than for Fas. In addition,
significantly higher levels of soluble FasL were found in marrow plasma of MDS
patients18. Soluble FasL seems to be functional in MDS as it inhibited the
growth of clonogenic CD34+/HLA-DR+ progenitors in a dose-dependant way78.
Similarly, suppression of apoptosis of BM mononuclear cells was observed by
treatment with anti-FasL75.
Overexpression (by RT-PCR and IH) of TNF? was detected more often than
overexpression of IFN? in BMMNC of MDS patients (79% vs. 42%,
respectively), in contrast to observations in normal BM54. The majority of TNF?
and IFN? producing cells were CD68+ macrophage lineage cells. TNF? and
IFN? upregulate Fas expression in a wide array of hematopoietic cells. A
significant correlation was found between TNF? protein levels in marrow
plasma and Fas expression on MDS marrow blasts18 and between TNF?
expression (by IH) and the extent of apoptosis in BM biopsies44.
The following model of immunoregulatory mechanisms in MDS can be
postulated from all these observations. Monoclonality develops as one of the
first hallmarks in early MDS. The immune system probably detects these
aberrant cells and an immune response is triggered. Activated CTL’s and NK-
cells show increasing expression of mFasL, whereas activated monocytes and
macrophages produce increasing amounts of TNF? and IFN?. Subsequently,
upregulation of Fas occurs, especially in the more mature cells. Massive
apoptosis develops in both normal and monoclonal compartment by enhanced
TNF? levels as well as by increased Fas/FasL interactions. Upregulated Fas-
bearing mature and immature normal and stromal BM cells die as innocent
bystanders and subsequently proliferation increases to compensate their loss.
As particularly proliferating myeloid progenitors have enhanced Fas
expression, they also die in increasing numbers (causing signal antonymy).
Upregulation of mFasL (subsequently leading to soluble FasL) is a way to
defend against attacking FasL-bearing CTL's, NK cells or macrophages. The
same happens to the rapidly dividing monoclonal cells, but as a consequence
of additional mutations/deletions during high mitotic pressure, these cells
acquire a differentiation defect and a survival benefit. Furthermore, these cells
are capable of turning down their Fas expression. Alternatively, they develop
non-functional truncated Fas splicing variants leading to a survival benefit and
consequently a growth advantage. On the other hand, as these blasts maintain
enhanced mFasL in order to escape from the triggered immune-surveillance,
their enhanced mFasL expression may also contribute to the increased killing
of polyclonal hematopoiesis and immunoregulatory cells with increased
expression of Fas. During MDS progression, evolution of leukemic clones with
decreasing Fas and increasing mFasL turn down their PCD machinery by
acquiring additional genetic aberrations. These leukemic clones progressively
develop growth advantage at the expense of increasing death of monoclonal
preleukemic and normal hematopoiesis.
General introduction ?27
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Apparent expansion of CD 34 Apparent expansion of CD 34+ + cells
during the evolution of myelodysplastic
syndromes to acute myeloid leukemia syndromes to acute myeloid leukemia
during the evolution of myelodysplastic
LFR Span, SE Dar, V Shetty, SD Mundle, L Broady-Robinson, S Alvi,
RAP Raymakers, TJM de Witte, A Raza
Background and objectives Background and objectives
Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the
primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the
proliferative characteristics of CD34+ cells in vivo of 33 untreated MDS patients (19 RA, 5 RARS, 7
RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML).
Materials and methods Materials and methods
All patients received a one hour infusion of the thymidine analogue iodo- or bromodeoxyuridine
intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry
technique by monoclonal anti-CD34 and anti-IUdR/BrdU antibodies was developed and performed.
By this technique we recognized CD34+ and CD34? cells actively engaged in DNA synthesis or not.
As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid
cells (mean value: RA/RARS 1.67%, RAEB(t) 8.68%, sAML 23.83%), as well as in the percentage
of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%, RAEB(t) 0.43% and sAML
3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI:
RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to
an exponential increase in the proportion of CD34+ cells of the proliferating compartment during
MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These
CD34+ cells appeared to proliferate more slowly than their more mature CD34? counterparts, since
we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during
MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 hours).
This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth
advantage leading to apparent expansion.
Expansion of CD34+ cells during the evolution of MDS to sAML?35
In myelodysplastic syndromes (MDS) the initial DNA-altering event probably
occurs at the level of the pluripotent stem cell. This early event may cause a
change in the cell cycle control mechanism that leads to a growth advantage of
this particular clone over their normal counterparts. This clonal expansion
causes the frequently found monoclonal hematopoiesis of the nonlymphoid
cells in MDS1-5. During this process of high proliferative activity secondary
events (DNA damaging events or DNA repair mechanism failures) induce a
cytogenetically marked subclone, like the frequently found 5q-, monosomy 7,
trisomy 8 clones in MDS6. This subclone or the following subclones7-9 are
characterized by even more complex cytogenetic abnormalities and are
recognized by their immaturity as blasts and/or as abnormally localised
immature precursors or ALIPs10-12. Eventually these subclones may cause the
evolution from RAEB(t) to acute myeloid leukemia (AML) by progressive
dedifferentiation and eventually losing their gene-directed programmed cell
death (PCD) or apoptosis13,14.
Over several years more evidence has been found that the high labeling index
(LI) in MDS is abrogated by a high apoptotic cell death9,14-16, which explains the
hypercellular bone marrow (BM) with peripheral cytopenias. This increased
apoptosis in MDS is visualized by different techniques like high/low molecular
weight DNA extraction by electrophoresis, in situ end labeling (ISEL) of DNA, in
situ terminal deoxynucleotidyl transferase (TUNEL) assay, and the Annexin V
assay by different research groups17-21. When RA/RARS develops to RAEB(t)
overall apoptosis is high. It slows down during the development of secondary
AML (sAML after previous MDS) because of the appearance of a progressive
number of ISEL-negative, immature myeloblasts. On the other hand, the high
myeloid LI decreases with slowing down of cell cycle times of myeloblasts
when evolution occurs to RAEBt and sAML9,13,15,22. It is still not proven whether
the high proliferation rate (mean overall myeloid LI of 25 to 30%) is a
compensatory mechanism for the high apoptosis rate in myelodysplasia9,23.
However, first results of treating MDS patients with pentoxifylline and
ciprofloxacin support this hypothesis: suppression of PCD (by turning down
transcription of TNF?) was followed by a decrease in overall myeloid LI24.
Very little is known regarding the specific proliferative characteristics of CD34+
cells in particular in MDS. By flow cytometry (FCM), a sufficient number of stem
cells after selection procedures can usually be obtained, but determining an
accurate LI from aspirated and separated cells is frequently proven to be
unreliable25,26. The best way of determining the accurate LI is obtaining BM
biopsies after in vivo labeling of S-phase cells13. Fortunately, we have been
able to develop an method to identify CD34+ cells and to simultaneously
examine their proliferative characteristics in BM biopsies. We conducted this
study to achieve a better understanding of their cycling properties during MDS
evolution and to study the process of a possible growth advantage of the clone
within CD34+ compartment, as more immature myeloblasts occur during MDS
progression to sAML.
Materials and methods
Thirty-three MDS patients (21 males and 12 females, mean (±SD) age 65.8
(±13.8) years) were studied for the proliferative characteristics of CD34+ cells.
All MDS patients (FAB classification:19 RA, 5 RARS, 7 RAEB and 2 RAEBt),
together with five sAML patients (four males and one female, mean age 52.8
(±10.4) years) and five “normals” (lymphoma patients with uninvolved BM, four
males and one female, mean age 56.8 (±22.7) years), who served as controls,
were eligible for study after informed consent was obtained. None of the
patients had received any therapy (except supportive care) for at least two
weeks prior to the one hour infusion of one of the thymidine analogues iodo- or
bromodeoxyuridine (IUdR/BrdU) at 100mg/m² intravenously, using a constant
rate infusion pump. Each infusion was immediately followed by a BM aspirate
and biopsy, which were handled on ice. The infusion protocols were reviewed
and approved by the Investigative Review Board of the Rush-Presbyterian-St.
Luke’s Medical Centre, National Cancer Institute (NCI) and the Food and Drug
Administration. The drugs were supplied by NCI. See Table 2.1 for detailed
individual characteristics of all patients and controls.
Single-labeling immunohistochemistry to measure overall myeloid
The BM biopsies were labeled by 3D9 (Bioscience Inc, Bethlehem, PA, USA)
antibodies against incorporated IUdR/BrdU to detect S-phase cells by showing
a brown punctation overlying their nucleï. After counterstaining and taking
myeloid morphology into account, an overall myeloid LI could be determined.
This procedure was described before in full detail27 and was also followed for
the sAML patients and controls.
Expansion of CD34+ cells during the evolution of MDS to sAML?37
Table 2.1 Proliferative characteristics of patients and controls expressed as a percentage of
three different compartments: total myeloid cells, total proliferating cells and total
CD34??cells (see Materials and methods section for details).
LI LICD34 CD34S/S
19.10 22.61 33.42 55.55 12.56 13.80 72.60
24.70 10.73 15.66 17.94 1.925
20.90 3.51 8.42 38.31 1.345 13.50 64.70
25.20 0.00 0.00 0.70 0.000
28.20 0.00 0.00 0.20 0.000
5.71 1.20 0.10 0.006
31.90 0.00 0.00 0.00 0.000
4.89 0.456 11.70 42.90
1.21 0.247 13.40 39.30
3.90 0.229 11.30 54.00
3.06 0.742 15.90 77.00
3.12 10.58 0.26 18.40 94.80
0.78 20.41 0.465
0.37 4.29 0.071
4.49 11.68 0.742
2.41 1.94 0.522
0.00 0.03 0.000
0.41 10.54 0.684 21.10 88.10
1.24 10.00 0.691 11.00 45.60
0.00 0.30 0.000
2.40 7.06 0.693
M=male, F=female, Ts and Tc in hours.
Double-labeling immunohistochemistry CD34/BrdU to measure
CD34 cells in S-phase or not
After fixation and decalcification of the BM biopsies, embedding in paraffin was
performed. Sections of approximately 6 ?m were placed on positively charged
Superfrost Plus slides, air-dried and used for immunohistochemistry (IH) at
room temperature. After deparaffinization by running through 100% xylene and
graded ethanols, rehydration in double distilled water (ddH2O) was followed by
incubation in freshly prepared 3% H2O2 for 30 minutes (min.) to block
endogenous peroxidase. After rinsing thoroughly in ddH2O, three washes in
respectively 0.15 M Tris Buffer Solution (TBS: 0.15 M sodium chloride in 0.05
M Tris buffer, pH 7.5) and 0.5 M TBS (0.5 M sodium chloride in 0.05 M Tris
buffer, pH 7.5) with 0.1% Tween 20 (Sigma)(TBST) was followed by a one hour
incubation with a monoclonal mouse anti-CD34 (dilution 1:10) antibody
(QBend/10, Biogenex). Again three washes in 0.5 M TBST which was followed
by 30 min. incubation with the secondary rabbit-antimouse IgG (1:20) antibody
Z259 (Dako, Carpinteria, CA, USA). After three washes in 0.5 M TBST, the
tertiary (1:40) antibody D651 (APAAP: alkaline phosphatase antialkaline
phosphatase complexes, Dako) was applied for 30 min., followed by washes in
0.5 M TBST. A blue color reaction was developed in the cell membrane and
cytoplasm by applying a freshly prepared BCIP/NBT (5-bromo-4-chloro-3-
indoxyl phosphate and nitro blue tetrazolium chloride, Dako) with 1 M
levamisole (1 ?l/ml) for approximately 3 to 10 min. by repeatedly checking the
blue color under the microscope. The BCIP/NBT reaction was neutralised by
rinsing in ddH2O. The slides were left overnight in 0.15 M phosphate buffer
solution (PBS) (0.15 M sodium chloride in 0.1 M phosphate buffer, pH 7.5) for
performing a second IH procedure the next day to detect cells in S-phase.
This procedure was started by applying 1mg/ml nuclease free pronase
(Calbiochem, LaJolla, CA, USA) for 45 min. incubation, followed by three
washes in 0.15 M PBS. 4N HCl treatment for 20 min. was done to permeabilize
cell- and nuclear membrane, which was followed by dip rinsing in ddH2O and
three washes in 0.15M PBS and 0.5 M PBST (0.5 M sodium chloride in 0.1 M
phosphate buffer with 0.1% Tween 20, pH 7.5) each. The incubation with the
primary anti-BrdU/IUdR (1:500) 3D9 containing 1.5% horse serum was stopped
after 60 min. with three washes in 0.5 M PBST. The secondary (1:200)
biotinylated monoclonal mouse antibody (Vectastain Elite Kit ABC, Vector,
Burlingham, CA, USA) in 1.5% horse serum was incubated for 30 min. After
washes in 0.5 M PBST, the tertiary (1:50) antibody ABC (avidin-
biotin/peroxidase complexes, Vectastain Elite Kit) was also incubated for 30
min. and followed by three washes in 0.5 M PBST. The brown color reaction
was developed in the nucleus with 50 mg DAB (3,3’-diaminobenzidine
tetrahydrochloride, Sigma) in 200 ml 0.05 M Tris buffer, pH 7.5, together with
12-14 ?l 30% H2O2 for 4 to 10 min., followed by three rinses in dd H2O.
Expansion of CD34+ cells during the evolution of MDS to sAML?39
Postfixation for the BCIP blue color was needed and performed with 2%
glutaraldehyde at 4ºC for 20 mins. Dehydration of these paraffin sections was
done by graded ethanols. After going through 100% xylene three times 30
seconds, the slides were mounted with xylene-based mounting solution
(Permount) using 2 ?m thick glass coverslips. No counterstain was used in this
Single-labeling immunohistochemistry to measure the percentage
of CD34+ cells of all myeloid cells
The same procedure after deparaffinization was followed as by the double-
labeling procedure, but we used DAB/ABC-kit to detect the CD34+ labeled cells
(brown cytoplasm and cell membrane) and counterstained the biopsy slides
with hematoxylin to differentiate CD34+ cells from the other myeloid cells. No
postfixation treatment was needed for these slides.
Morpho-immunohistochemical evaluation of BM biopsies:
detection and scoring of CD34+ cells and/or S-phase cells within
the myeloid compartment
At least 2000 positively labeled S-phase cells were counted from five or more
areas of the single-labeled biopsy for determination of the LI (%). Furthermore,
2000 S-phase labeled myeloid cells were counted by searching for CD34+
fields (aggregates or single cells) in the double-labeling method, whereas to
2000 myeloid cells were counted by searching for CD34+ cells in the single-
labeling method. Aggregates of three or more CD34+ cells were seen and
scored as a number of single cells. Erythroid and megakaryocytic cells were
excluded by morphology. This evaluation procedure was done twice (on
different days) by one and the same person, who is an experienced
hematomorphologist. These different cells were counted and expressed as a
percentage of one of the three different compartments (see Figure 2.1).
Autoradiography for determining Ts (duration of S-phase or DNA
doubling time) and calculation of Tc
The bone marrow aspirate samples were double-labeled in vitro with tritiated
thymidine for the calculation of the duration of S-phase (Ts) by our previously
described method13. With the use of Ts and LI the total cell cycling time Tc of
all myeloid cells can be calculated using the formula described by Wimber and
Quastler28: Tc=Ts ? GF/LI. GF is the growth fraction or the percentage of cells
in cycle, which was assumed to be 100%. Ts and Tc are both expressed in
hours in Table 2.1.
OVERALL MYELOIDOVERALL MYELOID OVERALL MYELOIDOVERALL MYELOIDOVERALL MYELOID OVERALL MYELOID
. . ... . . . . ............
. . ... . . . . ............
Figure 2.1 Overall myeloid compartment contains CD34+??and CD34? compartments, both contain
a proliferating fraction, taken together as S-phase compartment or LI of overall
L LI (%):
I (%):overall myeloid labeling index (LI) or percentage myeloid S-phase cells of all
myeloid cells in BM biopsies (grey area); L LICD34 (%):
cells of all CD34+ cells or the labeling index of CD34+ cells (spotted grey area divided
by spotted area); C CD34 (%):
D34 (%): percentage CD34+ cells of all myeloid cells (spotted
area); C CD34S/S (%):
D34S/S (%): percentage CD34+ cells in S-phase of all myeloid S-phase cells
(spotted grey area divided by grey area); C CD34S/M (%):
S-phase of all myeloid cells (%) can be calculated by multiplying CD34 (in %) by
LICD34 (in %) divided by 100 (spotted grey area divided by white area).
ICD34 (%): percentage CD34+ S-phase
D34S/M (%): percentage CD34+ cells in
After analyzing the distribution of the different continuous variables in the
various subgroups, only LI, Ts, and Tc showed a Gaussian distribution in all the
subgroups. Therefore, a distribution-free Wilcoxon-Mann-Whitney test was
used to determine statistical significant differences (P<0.05) between these
skewed-distributed subgroups (Table 2.2). Also the use of median values (with
25-75% interval) is statistically seen more correct to describe these parameters
when skewed distributions are involved. Pearson rank correlation tests were
used to analyse statistically significant correlations (P<0.05) between various
sets of two different variables within these subgroups.
After applying the double-labeling IH technique, CD34+ cells showed a blue
colored cytoplasm and cell membrane with variable intensity, while the S-phase
cells showed brown staining overlying the nucleus. CD34+ cells in S-phase are
therefore double-labeled (Figure 2.2). Aspecifically blue stained blood vessel
Expansion of CD34+ cells during the evolution of MDS to sAML?41
endothelium and collagen by QBend/10 was not taken into account but served
as positive internal control. The majority of cells were non-stained CD34? cells
not in S-phase. Sometimes CD34+ aggregates could be found in which all the
cells where in S-phase, especially in sAML and the more advanced stages of
MDS (Figure 2.3). The mean values (? standard error of mean; SEM) of all
determined proliferative parameters of all the groups are shown in Table 2.2
and Figures 2.4 and 2.5. Statistical differences between the various
(sub)groups and the level of significance in combination with the median values
(with 25-75% interval) of only the skewed-distributed parameters are expressed
in Table 2.2.
Table 2.2 Proliferative characteristics of the CD34+, S-phase and overall myeloid compartments
in controls, MDS subgroups and sAML.
Controls n=5 RA/RARS n=24
28.6 ± 1.38 25.8 ± 1.39
<0.1 9.60 ± 1.91
<0.1 0.35 ± 0.11 (b3, c3)
0.22 ± 0.12 1.67 ± 0.35 (a2, b1, c2) 8.68 ± 2.29 (d2)
0.1 (0.1–0.2) 1.03 (0.35–2.89)
CD34S/M (%) <0.1 0.189 (a2, b5, c3)
0.087 (0.035–0.247) 0.494 (0.260–0.684) 1.345 (0.693–1.93)
Ts (hours) 9.28 ± 1.02
Tc (hours) 39.8 ± 7.01
24.6 ± 1.91
6.29 ± 2.70
1.44 ± 0.52
21.5 ± 3.64
9.33 ± 3.87
11.98 ± 6.00 (e5)
23.83 ± 10.21 (e2)
10.5 ± 3.15
45.2 ± 15.5
11.0 ± 2.62
65.8 ± 3.68
Variables are expressed as mean percentage ±? SEM and median (25–75% interval, second line),
statistical significant differences between the following subgroups are designated as follows:
RA/RARS vs. controls (a), RA/RARS vs. RAEB(t) (b), RA/RARS vs. sAML (c), RAEB(t) vs. controls
(d), sAML vs. controls (e), the level of significance (P value) is expressed as a number in
superscript (5 means P<0.05 etc).
Labeling index of myeloid cells and CD34+ cells during MDS
A statistically non-significant decrease occurred in the mean overall myeloid LI:
RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5% (Table 2.2 and Figures 2.4
and 2.5). The controls/lymphoma patients with uninvolved BM had an
unexpectedly and unexplained high LI. Subsequently, we determined the size
of the CD34+ compartment and the LI of CD34+cells. A significant increase in
the mean percentage of CD34+ cells of all myeloid cells was found with the
progression from RA to sAML. The percentages CD34+ cells were 1.67% in
RA/RARS, 8.68% in RAEB(t) and 23.83% in sAML, much higher as compared
to 0.22% of the controls (Figure 2.4). On the other hand, no significant
differences were found in the LI of CD34+cells during MDS evolution. The
mean percentage of proliferating CD34+ cells within the CD34 compartment
was 9.60% in RA/RARS, 6.29% in RAEB(t) and 9.33% in sAML. However,
when the proliferating compartment of CD34+ cells as a fraction of all myeloid
cells (CD34S/M) was analysed, a significant exponential increase was found
during MDS evolution: 0.189% in RA/RARS, 0.429% in RAEB(t) and 3.304% in
sAML (Figure 2.4). An exponential increase of proliferating CD34+ cells as a
fraction of all myeloid proliferating cells (CD34S/S) was also seen with
progression of myelodysplasia to sAML: 0.35% in RA/RARS, 1.44% in RAEB(t)
and 11.98% in sAML (Figure 2.4). Sometimes we observed CD34+ aggregates
in which all the cells were in S-phase, especially in patients with a high CD34
expression (Figure 2.3).
Figure 2.3 Bone marrow biopsy of a patient with RAEBt showing a double-labeled (CD34+/BrdU+)
cell aggregate in which all the CD34+?cells are in S-phase.
Figure 2.2 Double-labeling immunohistochemistry CD34/BrdU in MDS bone marrow biopsy:
CD34+?cells show blue cytoplasm and cell membrane, whereas S-phase cells show a
brown nuclear staining. CD34???cells in S-phase have both features.
Expansion of CD34+ cells during the evolution of MDS to sAML?43
LI of total myeloid cells (%)
%CD34+ cells in S-phase of all myeloid S-phase cells (CD34S/S)
% CD34+ cells of all myeloid cells (CD34)
Control RA/RARSRAEB(t) sAML
Control RA/RARS RAEB(t)sAML
% CD34+ cells in S-phase of all myeloid cells (CD34S/M)
Figure 2.4 Proliferative characteristics of overall myeloid and CD34+??cells during MDS
These data suggest that the CD34+ compartment increases as MDS
progresses to sAML. Within this compartment the percentage proliferating
CD34+ cells remains the same (LICD34). The absolute number of proliferating
CD34+ cells (CD34S/M) increases therefore, whereas a decreasing trend
occurs in the size of the overall myeloid proliferating compartment (LI) as MDS
evolves to sAML. This means that within this decreasing proliferating overall
myeloid compartment, the fraction of proliferating CD34+ cells (CD34S/S)
increases at the expense of the proliferating CD34? fraction during MDS
evolution to sAML. The aforementioned changes in the different compartments
are depicted together in Figures 2.5a and 2.5b.
Figure 2.5 (a) Overall myeloid LI (%) divided in % proliferating CD34+??and CD34-????cells of all
myeloid cells during MDS progression to sAML. (b) Shift in size of CD34+ ?and CD34-
?compartment (as % of all myeloid cells) during MDS evolution to sAML.
Overall LI(%)Overall LI(%)
RA/RARS RA/RARSRAEB(t)RAEB(t) sAML sAML
RA/RARS RA/RARS RAEB(t)RAEB(t)sAML sAML
Duration of cell cycle phases
As MDS evolves to sAML, the mean total cell cycling time (Tc) increased
progressively, although it just missed statistical significance, whereas no
change occurred in the mean duration of S-phase (Ts). The mean Tc and Ts in
the different subgroups are respectively 39.8 and 9.28 hours for RA/RARS,
45.2 and 10.5 hours for RAEB(t), and 65.8 and 11.0 hours for sAML (Table 2.2
and Figure 2.6).
Correlations between various proliferative characteristics within
the different subgroups
Within the RA/RARS and RAEB(t) subgroups a strong significant correlation
was found between Tc and Ts, respectively r=0.84 (P<0.0004) and r=0.99
(P<0.0004), which was not found within the sAML group.
Expansion of CD34+ cells during the evolution of MDS to sAML?45
Figure 2.6 Mean total cell cycling time (Tc in hours, on top). ND, not determined.
Within the RA/RARS an increase in the CD34+ fraction of the total myeloid LI
leads to a longer Ts and to a longer overall Tc because the percentage
significantly positive correlated with Tc, Ts and CD34S/M (respectively r=0.59
(P<0.04), r=0.72 (P<0.008) and r=0.59 (P<0.005). Also CD34S/M was
significantly correlated with Tc and Ts, respectively r=0.74 (P<0.006) and
r=0.70 (P<0.01). The size of the CD34+ compartment depends on the
percentage of proliferating CD34+ cells of all myeloid cells: CD34 was
significantly correlated with CD34S/M (r=0.64, P<0.002). This means that
within the CD34+ compartment of the RA/RARS group the balance between
cell proliferation or cycling is favored over apoptosis .
Within the RAEB(t) group and the controls we did not find any of the
aforementioned correlations. Regarding the sAML group, similar characteristics
were found as in the RA/RARS group: a higher CD34+ fraction within the
overall myeloid LI leads to a longer overall Tc and an increment in the
percentage of proliferating CD34+ cells of all myeloid cells because the
percentage of proliferating CD34+ cells of all S-phase cells (CD34S/S) was
positively correlated with Tc (r=0.99, P<0.08) and CD34S/M (r=0.94, P<0.02).
Furthermore, the percentage CD34+ cells of all myeloid cells (CD34) was
positively correlated with CD34S/S and CD34S/M, respectively r=0.86 (P<0.06)
and r=0.83 (P<0.08). Only in sAML a positive correlation was found between
LICD34 and CD34S/M (r=0.90, P<0.04). This means that in sAML the balance
between CD34 cell proliferation or cycling and apoptosis is even more favored
for proliferation than it is the case in RA/RARS.
ControlControlRA/RARS RA/RARS RAEB(t)RAEB(t) sAMLsAML
+ cells of all myeloid S-phase cells (CD34S/S) was
The aim of this study was to get a better insight into the biology of CD34+ cells
in relation to the overall myeloid population during the evolution of MDS.
Therefore we investigated the evolutionary pattern of proliferation of myeloid
cells in general and CD34+ cells in particular by means of IH double-labeling on
BM biopsies. In patients with a low CD34 expression the CD34 parameters are
a little overrated because of searching for positive fields for CD34 expression.
This is not the case in patients with a high CD34 expression because of the
fact that almost every field could be scored. Despite some overinterpretation,
our values of CD34 (%) are within the same range as observed for the different
FAB classifications by a similar study of Soligo et al.29 using a different method
for scoring single-label CD34 immunohistochemistry in BM biopsies of MDS
During MDS progression we observed a significant increment in the percentage
of CD34+ cells of all myeloid cells (CD34), which means that the CD34+
compartment enlarges during progression from RA to sAML, which follows a
concomitant increase in the percentage of BM blasts by FAB classification.
Furthermore, as the overall myeloid LI shows a decreasing tendency from MDS
transition to sAML, the percentage proliferating CD34+ cells of all myeloid cells
(CD34S/M) increases exponentially at the expense of the proliferating CD34?
compartment. The LICD34 remains the same because the percentage S-phase
cells in the increasing CD34+ compartment remains the same, but the absolute
number of proliferating CD34+ cells in the CD34+ compartment increases and
consequently the absolute number of proliferating CD34+ cells of all myeloid
cells. This can be (partially) explained by an exponential increment in the
percentage of proliferating CD34+ cells of all myeloid cells in S-phase
(CD34S/S). Furthermore, a decreasing number of all myeloid cells in S-phase
concurrently with an increase in Tc and no change in Ts during MDS evolution
to sAML can be explained by the slower proliferation of the enlarged
proliferating CD34+ compartment. Immature CD34+ blasts must cycle slower
than their CD34? counterparts, otherwise a fast CD34+ overgrowth within the
BM would occur in every case of MDS, unless apoptosis or a rapid transit time
to the CD34? compartment would prevent this overgrowth. As MDS evolves
enhanced differentiation loss and decreased apoptosis occurs in the CD34+
compartment (especially as leukemic blasts are concerned) which further
increases the size of this compartment. From previous proliferation studies we
already know that during the evolution within MDS and to sAML the overall
myeloid LI decreases as Tc prolongs9,26, but it still has to be proven if (a
substantial number of) these increased CD34+ clonogenic blasts with a longer
duration of G0/G1-phase cause this phenomenon. Above all, the phenomenon
of “signal antonymy” (dying S-phase cells) in MDS was not taken into account
Expansion of CD34+ cells during the evolution of MDS to sAML?47
and this characteristic feature of MDS has to be investigated also for its
implications for measuring overall Ts and consequently Tc.
Myelodysplasia is primarily characterised by a phase of monoclonality before a
(rapid) expansion of leukemic blasts occur. Mehrotra et al.4 showed in AML that
the frequency of cytogenetically aberrant stem cells (CD34+lin?) is uncoupled
from compartment size, which means that additional mutation(s) and
maturation loss of the blast subpopulation is needed before expansion can
occur. Also in MDS these characteristics can be found30,31. Furthermore, it was
also found by interphase FISH analysis that the percentage of cytogenetically
aberrant cells in the CD34? compartment was higher than in the CD34+
compartment in good prognosis MDS, whereas this percentage of aberrant
cells was almost the same in both compartments in poor-prognosis MDS30.
Dynamic processes of clonal expansion and suppression of normal
hematopoiesis and the balance between them are involved in these
phenomena. These additional mutations may lead to apparent (and eventually
malignant) clonal expansion by several mechanisms: enhanced dedifferen-
tiation or differentiation arrest, increased autonomic proliferation, turning down
of apoptosis and eventually decreasing cell cycling times of clonogenic blasts.
These mechanisms are clearly incorporated in the FAB classification: the
progression of blasts, but even better in the IPSS score. Enhanced
dedifferentiation of MDS CD34+ cells was also found in in vitro cultures upon
growth factors32-34. No clear reports can be found investigating the increased
autonomic proliferation in MDS. In RAEB(t) and sAML, we previously showed
less overall apoptosis, especially in blasts, by using ISEL on BM biopsies14.
This phenomenon was also recently reported by Bouscary et al.35 by showing a
lower Fas expression on CD34+ cells of patients with advanced stages of MDS
and sAML when compared with early MDS stages, which was also associated
with less PCD by the TUNEL technique. In our study, we only found a strong
positive correlation between LICD34 and CD34S/M in sAML (r=0.90, P<0.04),
probably because apoptosis was found to be completely negative in this
clonogenic blast population14. Evidence for increased proliferation with slower
cell cycling times of CD34+ cells during MDS progression is also reported in
this article. The mechanism of eventually decreasing total cell cycling times is
seen in the blast subpopulation of ALIP-positive RAEB(t) patients with a very
fast evolution to sAML11,13.
The typical profile of exponential proliferation of CD34+ cells during MDS
evolution may be explained by a progressive autonomous proliferation in the
CD34+ compartment. Some evidence seen in our biopsies for a possible role of
paracrine-induced “signal synchronisation” in CD34+ aggregates, in which all
the CD34+ cells were in S-phase, could be used as a morphological substrate
for this autonomic proliferation (Figure 2.3). Increasing evidence for autocrine
and/or paracrine mechanisms for the explanation of the autonomous growth of
(and anti-apoptotic effects in) AML blasts is found in the literature today36,37, but
no studies in MDS are performed or reported as yet.
Different groups have already shown that the absolute percentage of CD34+
cells/blasts (CD34%) increases during the evolution of MDS38,39. The MDS
CD34+ cells show clearly promoted proliferative capacity (high cluster/colony
ratio in semi-solid medium) with strongly impaired differentiation upon growth
factors in in vitro cultures32-34. TNF? is one of the cytokines which may have a
dual role in this process: stimulation of proliferation of presumably early
progenitor cells and induction of apoptosis in their more mature
progeny9,14,20,40,41. TNF? directly stimulates the recruitment and proliferation of
very early, primitive progenitors (CD34++,CD38?) and induces an increased
resistance of the inhibitory effect of TGF? on these early stem cells40,42,43. The
immunohistochemically detected high TNF? levels in the BM biopsies of MDS
patients are therefore a likely explanation of the increment in the absolute
number of CD34+ cells, as well as the proliferating fraction of CD34+ cells when
MDS evolves9,14,41,44. On the other hand, a positive relationship between the
degree of PCD and the level of TNF? in the BM biopsies with a great
preponderance of TNF? specifically around more mature ISEL-positive cells
could explain the decrease in LI of CD34? cells, as well as a reduction in size of
the CD34? compartment found in this study. Of course the balance between the
various acting cytokines in the BM play the ultimate role in determining the
overall proliferation and apoptosis of myeloid cells.
CD34+ overexpression is observed in more than 30% of all MDS patients and
CD34 expression is higher (as percentage CD34+ single cells and CD34+
aggregates) in RAEB(t) than in RA/RARS29,32. The number of CD34+
aggregates in MDS biopsies are significantly positively correlated with the
percentage of BM blasts and ALIPs45. A significantly higher frequency of CD34
expression is also found in sAML (or therapy-related AML) when compared
with “de novo” AML46. In the present study, we showed a continuous process of
increasing CD34 expression during the evolution of myelodysplasia to sAML
caused by an increasing fraction of CD34+ cells within the total S-phase
population. CD34-positivity in BM and the presence of CD34+ cells in
circulation in MDS are both correlated with poor overall prognosis and with
leukemic transformation12,29,32,47,48. Within AML after MDS, this prognostic
relevance of CD34 expression has not been demonstrated yet, because no
studies on this selected subgroup within AML have been performed. In this
study we found a higher CD34 expression in AML after previous MDS as
compared with the MDS subgroups, which is the first evidence in literature of a
prognostic meaning of CD34 expression in AML-MDS. This poor prognostic
meaning of increasing CD34+ aggregates, as well as increasing CD34+
circulating cells in MDS can probably be explained by “signal synchronisation”.
This means that growth signalling or initiation in these blast cell aggregates
Expansion of CD34+ cells during the evolution of MDS to sAML?49 Download full-text
occurs in a paracrine fashion, causing these cells to go into S-phase at the
same time. This phenomenon is possibly responsible for autonomic growth by
overruling the need for stromal interactions for proliferation (Figure 2.3). These
speculations fit perfectly in the following model: when no or less stromal
interaction for growth purposes is needed, less activation of the cytokine-
dependent ?1-integrins very late antigen (VLA)-4 and VLA-5 on the CD34+ cells
can be assumed, which leads to a lower BM fibronectin adhesion and a higher
chance of circulating CD34+ cells49-51. Above all, recently a lower expression of
the cell adhesion molecule L-selectin was found in this primitive CD34+ cell
population of MDS patients compared to normals52. From a prognostic
perspective, it may therefore be very important to apply this double-labeling
immunohistochemistry to determine how many CD34+ cells and CD34+
aggregates can be found in the BM biopsies, and especially how many of them
are in S-phase.
S-Phase specific agents like cytosine arabinoside (Ara-C) are considered to be
most effective in MDS. If chronic cytoreductive therapy (like low-dose Ara-C) is
effective enough to kill (because Tc increases with no change in Ts) the
aberrant CD34+ stem cell clone(s) will be uncertain. New therapeutic options in
the growing population of MDS patients have to be developed. The
combination of cytoreductive therapy and biological therapy directed to
suppression of proliferation of the aberrant CD34+ clone(s) and suppression of
apoptosis of more differentiated BM cells may restore normal polyclonal
hematopoiesis in MDS patients.
In summary, as MDS evolves from low-risk to high-risk groups and eventually
sAML, we observed an increase in absolute CD34+ cells, as well as an
increase in absolute CD34+ cells in S-phase in BM biopsies. We believe that
this phenomenon is the result of clonal expansion of genetically altered blast
cells with less or no apoptosis and with slower proliferation rates than their
more mature CD34? counterparts in a different microenvironment of cytokines
and probably changed stromal interactions. The IH double-labeling technique
we described can be used to follow MDS evolution and probably determine
prognosis and leukemic transformation with greater accuracy than before and
irrespective of their FAB subtype, like Oriani et al.48 have shown for single-
labeling CD34 immunostaining. The different biological processes which lead to
changes in size and proliferative capacity of the CD34+ and CD34?
compartments within MDS in evolution have been discussed and are
pathophysiologically expressed in Figure 2.7.