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Leukemia Research 32 (2008) 1374–1381

Functional alterations of Lin−CD34+CD38+ cells in chronicmyelomonocytic leukemia and on progression to acute leukemia

Qian Sun a, Chi-Chiu So a, Sze-Fai Yip b, Thomas S.K. Wan b,Shiu-Kwan Ma a, L.C. Chan a,∗

a SH Ho Foundation Research Laboratories, Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong,Queen Mary Hospital, 102 Pokfulam Road, Hong Kong SAR, China

b Hematology Division, Department of Pathology, Queen Mary Hospital, Pokfulam, Hong Kong SAR, China

Received 25 October 2007; received in revised form 15 January 2008; accepted 13 February 2008Available online 26 March 2008

bstract

The functional behavior of hematopoietic stem cell (HSC) and progenitors in chronic myelomonocytic leukemia (CMML) and on diseaserogression is little known. We performed cell proliferation, apoptosis, hematopoietic colony forming/replating and differentiation potentialtudies in the purified subpopulations of Lin−CD34+CD38− and Lin−CD34+CD38+ cells from 16 CMML with 6 cases after acute myeloideukemia transformation (AML-t). We observed an expansion of the hematopoietic progenitor pool (Lin−CD34+ cells) in AML-t comprisingainly Lin−CD34+CD38+ cells. The Lin−CD34+CD38+ cells in AML-t displayed high proliferative activity, resistance to apoptosis, enhancedyeloid colony formation/replating ability and a complete dendritic cell (DC) differentiation block. Our findings suggest Lin−CD34+CD38+

− + −

ells instead of Lin CD34 CD38 cells could be the target(s) of secondary genetic lesions underpinning progression from CMML to AML,hich have implications for the further study of the biology of leukemic transformation and the design of new strategies for the effective

reatment of CMML.2008 Elsevier Ltd. All rights reserved.

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eywords: CMML; Proliferation; Apoptosis; Replating; Differentiation

. Introduction

Although leukemias are highly heterogeneous in terms ofhenotype, disease progression, prognosis, and response toherapy, the general mechanisms underlying leukemic trans-ormation are starting to be understood. Emerging evidencendicates that the cell origin of leukemia where the primaryhit’ or genetic alteration occurs can be in stem cells or theommitted progenitors whereas leukemia cells detected inhe patients (the tumor phenotype) reflect clonal expansionf cells as a result of secondary hits perturbing cellular andolecular pathways of self-renewal, proliferation, apoptosis

nd differentiation [1,2].Chronic myelomonocytic leukemia (CMML) as one of

he myelodysplastic/myeloproliferative syndromes has been

∗ Corresponding author. Tel.: +852 2855 3160; fax: +852 2817 7565.E-mail address: chanlc@pathology.hku.hk (L.C. Chan).

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145-2126/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2008.02.011

iewed as a clonal disorder of multipotent stem cell in ori-in [3–5]. CMML is characterized by the association ofonocytosis and granulocytosis, with a varying degree of

ysplasia and a frequency of transformation to acute myeloideukemia (AML) at 10–20% [6–10]. Previous studies onMML blasts from patients showed alterations in prolifera-

ion, apoptosis and colony forming ability [11–13]. However,MML blasts are phenotypically heterogeneous [14] and

hey comprise subpopulations with stem cell and/or progen-tor cells with little known about the role of each cellularompartment in contributing to the development of CMMLnd AML transformation. To address these issues, we haveurified Lin−CD34+CD38− and Lin−CD34+CD38+ sub-

opulations from patients in CMML phase and in acuteyeloid leukemia transformation (AML-t). Cell prolifera-

ion, apoptosis, hematopoietic colony forming and replatingbility were examined. In view of abnormalities of den-

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ritic cell (DC) differentiation in CMML [15] coupledith the increasing clinical interest in DCs as interest-

ng candidates for anti-tumor or anti-leukemic vaccinationtrategies, we also examined DC differentiation potentialrom these subpopulations in CMML and AML-t. Our find-ngs show that functional alterations are present mainlyn Lin−CD34+CD38+ subpopulations. Taken together, ouresults suggest Lin−CD34+CD38+ cells could be the targetsf secondary genetic lesions underpinning progression fromMML to AML.

. Materials and methods

.1. CMML patients and healthy controls

Sixteen patients with CMML who were registered in our insti-ute in the period 2003–2006 were studied (Table 1). Six of these 16ases (CMML(B)) progressed to AML (median time 10 months;ange 4–22 months). They had a higher mean blast count andore frequent cytogenetic abnormalities compared to the stable

roup (CMML(A)). This is in agreement with previous findingshat increased blast cell percentage and/or presence of cytogeneticbnormalities in CMML are more frequently associated with AMLvolution and poor prognosis [9,16]. Diagnosis of CMML andML-t was based on peripheral blood (PB) and bone marrow (BM)

xamination according to WHO classification. Twelve healthy vol-nteers from Beijing Blood Center (median age 45 years, range4–60) served as normal controls. Samples of BM or mobilized PBollowing administration of granulocyte colony stimulating factor

G-CSF) were obtained after written informed consent. All stud-es were conducted following the regulations of the institutionaleview board of University of Hong Kong/Hong Kong West Clusterospitals and the National Institute of Health Guide in China.

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able 1linical features of CMML patients

atients Age (year) WBC (×109 l−1) Monocytes (×MML without AML transformation, CMML(A)

1 71 12.5 2.22 72 50.1 3.93 81 32.1 2.64 81 67.2 6.15 70 35.8 3.06 72 43.2 3.57 81 41.5 4.38 76 14.9 1.69 83 18.9 1.4

10 70 22.3 2.5

MML with later AML transformation, CMML(B)a

11b 63 23.6 (138) 1.4 (3.7)12 72 26.3 (7.5) 1.8 (2.1)13b 80 34.7 (5.4) 2.4 (1.0)14b 56 57.0 (237.1) 9.3 (16.9)15 73 62.3 (25.7) 6.7 (7.8)16b 44 30.1 (113) 3.5 (23.4)

BC, white blood cell count; BM, bone marrow; ND, not done.a Median time from diagnosis to AML transformation is 10 months, range 4–22 mb CMML-2 according to WHO classification 2001. All others belong to CMML-

h 32 (2008) 1374–1381 1375

.2. Cell preparation

BM or PB mononuclear cells (PBMC) were isolated by density-radient centrifugation (Ficoll 1.077, Sigma, USA) and Lin− cellsere obtained through negative selection using Lineage Cell Deple-

ion Kit (Miltenyi Biotech, Germany) followed by positive selectionf CD34+ cells from Lin− cells using CD34 MicroBead Kit (Mil-enyi Biotech, Germany) according to manufacturer instructions.he purity of Lin−CD34+ cells was greater than 92%. Lin−CD34+

D38− and Lin−CD34+CD38+ fractions were further sorted outrom the above-enriched Lin−CD34+ cells using an Epics-Altra cellorter by staining with PE-conjugated anti-CD38 mAbs (BD Bio-ciences, San Diego, USA). The purity of Lin−CD34+CD38− andin−CD34+CD38+ fractions was >95%. We included CD38low andD38high subpopulations together as CD38+ cells in this study, inoncordance with many previous studies [17–19].

.3. Cell proliferation, apoptotic assays

Purified Lin−CD34+CD38− and Lin−CD34+CD38+ cells weresed in the studies of proliferation, apoptosis and colony formingssay. To measure proliferative activity, cells were pre-stimulatedith a cytokine cocktail including 100 ng/ml stem cell factor (SCF),0 ng/ml Flt3 ligand (FL) and 10 ng/ml IL-3 (R&D, Hamberg, Ger-any) for 48 h in complete IMDM medium and pulsed with 0.5 �Ci

H thymidine for an additional 18 h and thymidine incorporationas measured by a scintillation counter. To investigate cell cycle

tatus at the S-G2/M phase, cells were stimulated with the aboveytokines for 48 h, harvested and fixed in 70% (v/v) ethanol. Theells were then stained with 20 �g/ml propidium iodide (PI) and0 �g/ml ribonuclease A (Sigma, USA) for 20 min at 37 ◦C and

hen analyzed by flow cytometry.

To measure TNF alpha-induced apoptotic activity, 5 × 104 cellsere cultured in complete IMDM medium in 96-well plate for4 h consisting of 100 ng/ml TNF alpha (R&D, Hamberg, Ger-any) instead of additional cytokines, then immediately stained

109 l−1) BM blasts (%) Cytogenetics

2 46,XY [9]1 46,XX [10]5 46,XY [10]4 46,XY [11]2 46,XX [11]3 46,XY [10]8 ND5 45,Y,-X [12]3 ND6 ND

10 (40) 46,XX,t(5;12) (q34;q24) [15]3 (28) ND

10 (30) 46,XY [12]12 (32) 47,XX,+8 [22]6 (40) 46,XY [14]

15 (46) 46,XY,t(3;3) (q21;q26) [15]

onths. Data in parenthesis represent counts after AML transformation.1.

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ith FITC-conjugated annexin V and PI (BD Biosciences, Saniego, USA) for 15 min at room temperature in the dark. The per-

entage of apoptotic cells, i.e. annexin V+ and PI−, were determinedy FACS Calibur within 5 min of staining.

.4. Hematopoietic colony forming and replating assay

Purified Lin−CD34+CD38− and Lin−CD34+CD38+ cells capa-le of forming colonies (colony forming unit, CFU) were assayedn methylcellulose-based semisolid cultures consisting of hSCF,EPO, hGM-CSF, hIL-3 (MethoCult GF H4434, StemCell Tech-ologies Inc., Vancouver, Canada). Cells were plated at a finaloncentration of 1 × 103 cells/ml in 35 mm culture dish and cul-ured for 14 days (CFU-E for 7 days). To assess replating potential,ells derived from individual colonies at day 14 were harvestednd replated in 96-well plate at a 14-day interval. Hematopoi-tic colonies were identified by inverted microscope as previouslyescribed [20]: CFU-Mix (colonies containing both erythroidnd myeloid cells); CFU-erythroid (erythroid clusters of 20-50emoglobinized cells (CFU-E) and erythroid colonies of >50emoglobinized cells grouped in one or several clusters (BFU-E)),FU-GM (myeloid colonies comprised the identifiable subpopula-

ions of >40 granulocytes and macrophages).

.5. DC generation and flow cytometry

− + − − + +

Purified Lin CD34 CD38 and Lin CD34 CD38 cells werexamined for DC differentiation abilities in complete mediumonsisting of RPMI 1640, 10% FBS in 48-well culture plate, supple-ented with 50 ng/ml GM-CSF, 10 ng/ml IL-3 and 10 ng/ml TNF

lpha (R&D, Hamberg, Germany). Cells were cultured at 37 ◦C in

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ig. 1. Percentage of Lin−CD34+ pool and Lin−CD34+CD38+ compartment in Cononuclear cell fraction and the proportion of Lin−CD34+CD38+ cells within L

ncrease in Lin−CD34+ cells and Lin−CD34+CD38+ cells is seen both in PB and BMCMML(A), n = 5), and the subgroup of CMML(B) before (Pre-AML-t, n = 6) and

h 32 (2008) 1374–1381

humidified 5% CO2 containing atmosphere. At day 10, cells werearvested for immunophenotype analysis.

For immunophenotyping of DCs, cells from day 10 culturesere stained with a cocktail of lineage antibodies conjugatedith FITC against CD3, CD14, CD19, CD20 and CD56, PerCP-

onjugated CD11c, PE-conjugated HLA-DR, CD80, CD86 withatched isotype controls (BD Biosciences, USA). In most cases× 104 live cells gated on PI exclusion were collected for analy-

is. Lin−CD11c+HLA-DR+ cells were defined as DCs. Cells werecquired by FACS Calibur using CellQuest 3.1 (BD Biosciences,an Diego, USA). Staining intensities were expressed as arithmeticean fluorescence intensity (MFI) calculated using CellQuest 3.1.

.6. Statistical analysis

Statistical analysis was performed by one-way analysis of vari-nce (ANOVA) with Bonferroni correction using the SPSS softwareackage to examine the differences within the means of the nor-al and CMML groups. In addition, Student’s t-test was used

n comparison between the subgroups of CMML(B) before andfter transformation. Results were presented as the mean ± S.E.M.,nless otherwise indicated.

. Results

.1. Expansion of Lin−CD34+CD38+ pool in CMML on

rogression to AML

We first examined the total population of Lin−CD34+

ells in mobilized PB after G-CSF administration as well

MML and on progression to AML. The percentage of Lin−CD34+ cells inin−CD34+ fraction in mobilized PB (A) or BM (B) are shown. Significantof AML-t group. Normal (n = 12), CMML without leukemia transformationafter AML transformation (AML-t, n = 6).

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s the proportion of Lin−CD34+CD38+ cells within thenriched Lin−CD34+ cells from the following subgroups:MML without leukemia transformation in the studyeriod (CMML(A), patients 1–10); a subgroup of CMMLCMML(B), patients 11–16) analyzed both before (Pre-ML-t) and after transformation (AML-t). We observed an

ncrement of Lin−CD34+ cells only in AML-t (3.0 ± 0.25%)hen compared with normal (1.85 ± 0.35%). We foundo significant difference in Lin−CD34+ cell percentagen CMML(A) or Pre-AML-t when compared with normalontrol (P > 0.05) (Fig. 1A). In consistent with some sub-ypes of de novo AML (M4, M5) [21–23], we did notbserve a very high percentage of Lin−CD34+ cells in PBn AML-t (3.0 ± 0.25%). We observed a similar pattern inM samples in CMML and on progression to AML withonsiderably increased Lin−CD34+ cells in AML-t com-ared with the other groups (5–10-fold, P < 0.01) (Fig. 1B).

urthermore, we showed that AML-t Lin−CD34+ cells com-rised more CD38+ progenitors than CD38− cells, indicatingn expansion of Lin−CD34+CD38+ compartment in AML-tP < 0.05).

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ig. 2. Proliferative activity of Lin−CD34+CD38+ cells and Lin−CD34+CD38−n S-G2/M phase after cytokine stimulation. (A) Cell proliferation was measuredlled bars/circles indicate Lin−CD34+CD38− and Lin−CD34+CD38+ cells, resin−CD34+CD38+ cells is seen from CMML on progression to AML-t. (B) Hisf Lin−CD34+CD38− and Lin−CD34+CD38+ cells in S-G2/M phase after cytokinn S-G2/M phase is only seen in the Lin−CD34+CD38+ subgroup. Comparisonsre-AML-t and AML-t. Normal (n = 8), CMML(A) (n = 5), Pre-AML-t (n = 6) and

h 32 (2008) 1374–1381 1377

.2. Lin−CD34+CD38+ cells showed increased cellroliferation and increased S-G2/M status onrogression to AML

We performed the 3H-thymidine uptake abilities of puri-ed Lin−CD34+CD38− and Lin−CD34+CD38+ cells afterytokine stimulation from normal, CMML without leukemiaransformation (CMML(A)), Pre-AML-t CMML and AML-tFig. 2A). Lin−CD34+CD38+ cells from all CMML groupshowed increased proliferation compared with normal con-rol (P < 0.05), with the highest proliferative activity inhe subgroup of AML-t (P < 0.05). In contrast, no sig-ificant differences in proliferative activity was seen inin−CD34+CD38− cells from all CMML subgroups com-ared with normal (P > 0.05). Since HSC is a quiescentopulation with a small proportion actively entering cell cycle24], we speculated that the increased proliferation as evident

y increased thymidine incorporation reflected the increasedropensity of Lin−CD34+CD38+ cells to enter cell cycle.his was confirmed on analysis of cell cycle status whichhowed an increase in the percentage of cells in S-G2/M

cells as measured by 3H thymidine incorporation and percentage of cellsby a scintillation counter shown as counts per minute (CPM). Open andpectively. A significant and progressive increase in cell proliferation intogram plots and (C and D) graphical representations of the percentagee stimulation were measured by PI staining. A significant increase of cellsare made between CMML(A)/Pre-AML-t and normal as well as betweenAML-t (n = 6).

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hase in Lin−CD34+CD38+ cells in CMML before and aftereukemia transformation, with the highest percentage seen inML-t (P < 0.01) (Fig. 2D) with a representative histogram

n Fig. 2B. In contrast, we did not detect any significant differ-nce in percentages of Lin−CD34+CD38− cells in S-G2/Mhase among normal control, CMML(A), Pre-AML-t andML-t (P > 0.05) (Fig. 2C).

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ig. 3. TNF alpha-induced apoptotic activities of Lin−CD34+CD38+ and Lin−in−CD34+CD38− and Lin−CD34+CD38+ cells after 24 h treatment with TNF alp

B) Graphical representation of the percentage of annexin V+ and PI− cells in eaompartment, where an increase in TNF alpha-induced apoptosis is observed in CMdecrease in apoptosis in AML-t with or without TNF alpha treatment. Normal (n

h 32 (2008) 1374–1381

.3. Lin−CD34+CD38+ cells showed resistance topoptosis, enhanced myeloid colony growth andeplating capacity on progression to AML

In view of previous report of co-existence of increased pro-iferation and apoptosis in CMML [11,12], we investigatedNF alpha-induced apoptotic activity of Lin−CD34+CD38−

CD34+CD38− cells. (A) FACS plots showing apoptosis from purifiedha, with untreated cells incubated with medium only served as the control.ch group. Significant differences are seen only in the Lin−CD34+CD38+

ML cases (CMML(A) and Pre-AML-t) when compared with Normal, and= 5), CMML(A) (n = 8), Pre-AML-t (n = 6) and AML-t (n = 6).

Q. Sun et al. / Leukemia Research 32 (2008) 1374–1381 1379

Table 2Colony forming ability of Lin−CD34+CD38− and Lin−CD34+CD38+ cells

Group Lin−CD34+CD38− Lin−CD34+CD38+

CFU-Mix CFU-GM Erythroid CFU-Mix CFU-GM Erythroid

Normal (n = 5) 5 ± 3 43 ± 8 47 ± 18 BD 51 ± 17 8 ± 5CMML(A) (n = 4) 3 ± 1 38 ± 6 27 ± 9a BD 63 ± 6a BDPre-AML-t (n = 5) 4 ± 1 40 ± 7 20 ± 6a BD 68 ± 11a BDA 26 ±R

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tCDtaDwAA(atPDoLLin−34+CD38+ and Lin−CD34+CD38− cells in CMML(A),Pre-AML-t and barely detected from Lin−34+CD38+ cells inAML-t (data not shown).

Fig. 4. Myeloid colony forming and replating capacity ofLin−CD34+CD38− and Lin−CD34+CD38+ cells. The efficiency ofmyeloid colony formation and replating capacity were determined by thenumbers of colonies per 103 plating/replating cells. The data clearly showsa significant and gradual increase in myeloid colony formation in theLin−CD34+CD38+ fraction from normal to CMML(A) through to AML-t.

ML-t (n = 5) 3 ± 2 37 ± 9

esults represent mean number of colonies per 103 plating cells ± S.D.a Significantly different from normal control (P < 0.05); BD: below detec

nd Lin−CD34+CD38+ cells. A representative sample inach subgroup measured by FACS was shown in Fig. 3A.e observed elevated levels of TNF alpha-induced apop-

osis and cell death in Lin−CD34+CD38+ cells in theubgroups of CMML without or before leukemia transfor-ation (CMML(A) and Pre-AML-t) as compared to normal

∼1-fold, P < 0.01). In contrast, Lin−CD34+CD38+ cells inML-t showed significantly reduced apoptosis in spite ofNF alpha induction, as shown in Fig. 3B (P < 0.01). We next

nvestigated the colony forming capacity of the Lin−CD34+

ells as measured by the number of colonies per 103 plat-ng cells (Table 2). A statistically significant increase inhe numbers of CFU-GM derived from Lin−CD34+CD38+

rogenitors was observed in all CMML subgroups whenompared with normal control, with the highest myeloidolony formation in AML-t subgroup (P < 0.05). No signif-cant alteration of myeloid colony formation was found inin−CD34+CD38− compartment in all CMML subgroupshen compared with normal. CFU-Mix multipotent progeni-

ors were slightly decreased (P > 0.05) in Lin−CD34+CD38−ells among all the subgroups of CMML and barely detectedn Lin−CD34+CD38+ progenitors. A significantly reducedrythroid colonies in all CMML patients was observed inoth Lin−CD34+CD38− and Lin−CD34+CD38+ popula-ions (P < 0.05).

We hypothesized that committed myeloid progeni-ors could be the targets of leukemic transformingvents through the acquisition of self-renewal property inin−CD34+CD38+ cells. We next examined the replat-

ng ability of cells derived from each individual coloniess a surrogate assay to evaluate self-renewal in vitro aseported in previous studies [18,25]. Within the limitsf this study, we examined short-term replating potential.nly Lin−CD34+CD38+ cells from AML-t showed myeloid

olony formation with replating ability. This contrastedith the general ability of Lin−CD34+CD38− cells to form

olonies and replate among all groups, with no significantifference among them (P > 0.05), as shown in Fig. 4. Takenogether, these results provide evidence linking increasedesistance to apoptotic signal as well as enhanced myeloidolony growth/replating property of Lin−CD34+CD38+ cells

n progression to AML. Whether Lin−CD34+CD38+ cellsave acquired the self-renewal property of stem cell on AMLrogression can be explored in future studies of long-termepopulation assays.

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.4. Both Lin−CD34+CD38− and Lin−CD34+CD38+

ells displayed DC differentiation block

We next evaluated in vitro DC differentiation poten-ial of Lin−34+CD38− and Lin−CD34+CD38+ cells inMML. Under the culture condition permissive forC generation, cells were cultured for 10 days and

he frequency of DCs was determined by the percent-ge of Lin−CD11c+HLA-DR+ cells by flow cytometry.C differentiation potential in Lin−CD34+CD38− cellsas reduced in CMML(A) (40.3 ± 4.0%, P < 0.05), Pre-ML-t (41.5 ± 3.6%, P < 0.05), and severely reduced inML-t (16.7 ± 4.3%, P < 0.01) as compared with normal

59.7 ± 4.0%) (Fig. 5A). Lin−34+CD38+ cells in CMML(A)nd Pre-AML-t also displayed reduced DC differentia-ion compared to normal (50–52 ± 5.1% vs. 62.3 ± 6.5%,< 0.05). The reduction was striking in AML-t withCs barely detected (Fig. 5B). Similarly, the expressionf co-stimulatory molecules (CD80, CD86) within thein−CD11c+ DC gate was reduced in DCs derived from both

nly Lin−CD34+CD38+ cells in the AML-t group show measurableeplating potential. A significant difference in myeloid colony formation oreplating efficiency in the Lin−CD34+CD38− cells among various groupss not obvious. Normal (n = 5), CMML(A) (n = 4), Pre-AML-t (n = 5) andML-t (n = 5).

1380 Q. Sun et al. / Leukemia Research 32 (2008) 1374–1381

Fig. 5. Induction of DC differentiation from Lin−CD34+CD38− and Lin−CD34+CD38+ cells. DCs derived from Lin−CD34+CD38− cells (A) andL HLA-Dd −CD34t t (n = 6

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rops in DC differentiation are seen in both Lin−CD34+CD38− cells and Lino AML-t. Normal (n = 6), CMML(A) (n = 5), Pre-AML-t (n = 6) and AML-

. Discussion

Our studies into the functional analysis of Lin−34+CD38−nd Lin−CD34+CD38+ subpopulations in CMML wereased on observations that circulating human hematopoi-tic stem cells (HSC) mobilized from BM using G-CSFre Lin−CD34+CD38− cells [26], whereas in transplanta-ion studies of human leukemia cells into SCID mice thelonogenic or self-renewal population (leukemic stem cells)an arise from Lin−CD34+CD38− cells from human AML27] and from granulocyte–marophage progenitors, a sub-opulation of Lin−CD34+CD38+ cells in patients with CMLlast-crisis [18]. Thus functional characterization of differ-nt hematopoietic compartments in leukemia patients shouldrovide insights into the biology of leukemic transformation.

In the present study, we showed an expansion ofin−CD34+ pool in CMML only after transformation

AML-t) and that is mainly due to an increase ofin−CD34+CD38+ cells. Functional analysis of subpopula-

ions of Lin−CD34+ pool revealed that Lin−CD34+CD38+

ells, but not Lin−CD34+CD38− cells, exhibit a progressivencrease in proliferation potential and myeloid colony for-

ation from CMML to AML-t. This is in concordance withrevious studies showing CMML patients with high sponta-eous CFU-GM colony growth are associated with a shorterurvival and a tendency to transform into AML-t [28,29].ur study also demonstrated that the Lin−CD34+CD38+

ells, but not the Lin−CD34+CD38− cells in CMML acquirerelative resistance to TNF alpha-induced apoptosis andyeloid colony replating ability on progression to AML.ince myeloid progenitors have limited or no self-renewalapacity demonstrated in long-term culture initiating-cellLTC-IC) assays by Weissman’s group [19], whether thesein−CD34+CD38+ cells have acquired self-renewal abilityn AML progression should be validated with LTC-IC assayo evaluate leukemic differentiation program along with thebility to produce engraftment in SCID mice in future studies.

Our overall findings are in line with a study on CMLn blast-crisis, which demonstrated that changes in myeloidolony formation and replating ability occur within thein−CD34+CD38+ compartment rather than the upstream

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R+ cells by flow cytometry at culture of day 10. Significant and progressive+CD38+ cells from normal to CMML (CMML(A) and Pre-AML-t) through

).

in−CD34+CD38− compartment [18]. The lack of demon-trable changes in myeloid colony formation and replatingbility in the Lin−CD34+CD38− cells may indicate that theyre not the target for secondary hits causing blastic trans-ormation. Alternatively, they may just represent residualormal Lin−CD34+CD38− cells in these patients. In thisegard, it will be interesting to know the cytogenetics of theematopoietic colonies grown from this Lin−CD34+CD38−ells. Unfortunately, experimental materials from sorted cellsere not saved for cytogenetic analysis.We also observed progressive impairment in DC differen-

iation from CMML to AML-t, the degree of which is mostrominent in the Lin−34+CD38+ compartment. The findingf impaired DC differentiation in the Lin−CD34+CD38−ells may argue against them being residual normal cellsn these patients while the more pronounced impairmentn DC differentiation in the Lin−34+CD38+ compartment

ay support the notion they are the target for secondaryhanges leading to clonal evolution and leukemic transfor-ation. However, one cannot exclude the possibility that the

bserved abnormality in DC differentiation is not intrinsico the leukemic cells but reflects a failure of the immuneystem accompanying leukemic progression. The answer tohis question is not only of biologic interest but also of clin-cal relevance in view of increasing research into anti-tumormmunity and DC-based vaccines.

In conclusion, results of this study provide evidencehat functional alterations of Lin−CD34+ cells in CMMLre mainly present in Lin−CD34+CD38+ compartment.otwithstanding the limitations of our clinical samples, ourata suggest that Lin−CD34+CD38+ cells are the likelyellular target of leukemic transformation in CMML. Thisontrasts with previous studies of de novo AML showinghat Lin−CD34+CD38− cells have decreased Fas-inducedpoptosis, reduced immunogenicity and impaired DC trans-ormation capacities [17]. We believe further studies ofin−CD34+CD38+ cells as well as Lin−CD34+CD38− cells

y transplantation studies in SCID mice will help furthero identify the phenotypic and functional characteristics ofeukemia stem cells in CMML. Results from these stud-es will certainly provide key insights into the biology of

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eukemia transformation and facilitate more efficient drugesign and therapy.

onflict of interest

There is no conflict of interest to be disclosed.

cknowledgements

We thank Mr. Cary So for his excellent technical supportnd Ms. Juliana Kwok for manuscript preparation.

eferences

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