Effect of lenalidomide therapy on hematopoiesis of patients withmyelodysplastic syndromes associated with chromosome 5qdeletion

by Maria Ximeri, Athanasios Galanopoulos, Mirjam Klaus, Agapi Parcharidou,Krinio Giannikou, Maria Psyllaki, Argyrios Symeonidis, Vasiliki Pappa,Zafiris Kartasis, Dimitra Liapi, Eleftheria Hatzimichael, Styliani Kokoris,Penelope Korkolopoulou, Constantina Sambani, Charalampos Pontikoglou,and Helen A. Papadaki

Haematologica 2009 [Epub ahead of print]

doi:10.3324/haematol.2009.010876

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Copyright 2009 Ferrata Storti Foundation.Published Ahead of Print on September 22, 2009, as doi:10.3324/haematol.2009.010876.

Original Article

Funding: the study wassupported by a grant from theHellenic General Secretary ofResearch and Technology(PENED # 03__72).

Manuscript received onMay 2, 2009; revisedversion arrived on July 23,2009; manuscript accepted onAugust 6, 2009.

Correspondence: Helen A Papadaki, MD, PhD,Professor of Haematology,Department of Hematology,University Hospital of Heraklion,P.O. Box 1352, Heraklion, Crete,Greece. E-mail:epapadak@med.uoc.gr

BackgroundLenalidomide improves erythropoiesis in patients with low/intermediate-1 risk myelodys-platic syndrome (MDS) and interstitial deletion of the long arm of chromosome 5 [del(5q)].The aim of this study was to explore the effect of lenalidomide treatment on the reservesand functional characteristics of bone marrow (BM) hematopoietic progenitor/precursorcells, BM stromal cells and peripheral blood (PB) lymphocytes in low/intermediate-1 riskMDS patients with del(5q).

Design and MethodsWe evaluated the number and clonogenic potential of BM erythroid/myeloid/megakary-ocytic progenitor cells using clonogenic assays, the apoptotic characteristics and adhesionmolecule expression of CD34+ cells by flow cytometry, the hematopoiesis supportingcapacity of BM stromal cells using long-term BM cultures and the number and activationstatus of PB lymphocytes in 10 low/intermediate-1 risk MDS patients with del(5q) receiv-ing lenalidomide.

ResultsCompared to baseline, lenalidomide treatment significantly decreased the proportion ofBM CD34+ cells, increased the proportion of CD36+/ClycoA+ and CD36–/GlycoA+ ery-throid cells and the percentage of apoptotic cells within these cell compartments.Treatment significantly improved the clonogenic potential of BM erythroid, myeloid,megakaryocytic colony-forming cells and increased the proportion of CD34+ cells express-ing the adhesion molecules CD11a, CD49d, CD54, CXCR4 and the SLAM antigen CD48.The hematopoiesis supporting capacity of BM stroma improved significantly followingtreatment as was demonstrated by the number of colony-forming cells and the level ofstromal-derived factor-1_ and intercellular adhesion molecule-1 in long-term BM culturesupernatants. Lenalidomide treatment also increased the proportion of activated PB T-lym-phocytes.

ConclusionsThe beneficial effect of lenalidomide in lower risk MDS patients with del(5q) is associat-ed with significant increase in the proportion of BM erythroid precursor cells and in thefrequency of clonogenic progenitor cells, substantial improvement in the hematopoiesissupporting potential of BM stroma and with significant alterations in the adhesion profileof BM CD34+ cells.

Key words: lenalidomide, hematopoiesis, MDS, chromosome 5q deletion.

Citation: Ximeri M, Galanopoulos A, Klaus M, Parcharidou A, Giannikou K, Psyllaki M,Symeonidis A, Pappa V, Kartasis Z, Liapi D, Hatzimichael E, Kokoris S, Korkolopoulou P,Sambani C, Pontikoglou C and Papadaki HA on behalf of the Hellenic MDS Study Group.Effectof lenalidomide therapy on hematopoiesis of patients with myelodysplastic syndromes associatedwith chromosome 5q deletion Haematologica 2009; doi:10.3324/haematol.2009.010876

©2010 Ferrata Storti Foundation. This is an open-access paper.

Effect of lenalidomide therapy on hematopoiesis of patients with myelodysplasticsyndromes associated with chromosome 5q deletion

Maria Ximeri,1 Athanasios Galanopoulos,2 Mirjam Klaus,1 Agapi Parcharidou,3 Krinio Giannikou,1 Maria Psyllaki,1

Argyrios Symeonidis,4 Vasiliki Pappa,5 Zafiris Kartasis,6 Dimitra Liapi,7 Eleftheria Hatzimichael,8 Styliani Kokoris,9

Penelope Korkolopoulou,10 Constantina Sambani,11 Charalampos Pontikoglou,1 and Helen A. Papadaki1 on behalf of

the Hellenic MDS Study Group

1Department of Hematology, University of Crete School of Medicine, Heraklion, Greece; 2Department of Clinical Hematology,“G.Gennimatas” Hospital, Athens, Greece; 3Third Department of Internal Medicine, Red Cross Hospital “Korgialenio Benakio”,Athens, Greece; 4Stem Cell Transplantation Unit, Department of Internal Medicine, Hematology Division, University of PatrasMedical School, Patras, Greece; 5Hematology Unit, Second Department of Internal Medicine-Propaedeutic, Attikon UniversityGeneral Hospital, University of Athens, Athens, Greece; 6Department of Hematology, General Hospital of Chalkis, Chalkis, Greece;7Department of Hematology, “Venizeleion” Hospital, Heraklion, Greece; 8Department of Hematology, University of Ioannina,Ioannina, Greece; 9Department of Hematology, National Kapodistrian University of Athens, Laikon University Hospital, Athens,Greece; 10First Department of Pathology, National and Kapodistrian University of Athens, Laiko Hospital, Athens, Greece;11Laboratory of Health Physics & Environmental Hygiene, Inst. NT-RP, National Center for Scientific Research (NCSR) 'Demokritos',Athens, Greece

ABSTRACT

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Introduction

Myelodysplastic syndromes (MDS) comprise a het-erogeneous group of hematopoietic stem cell malignan-cies characterized by ineffective bone marrow (BM)hematopoiesis, peripheral blood (PB) cytopenias andsubstantial risk for progression to acute myeloidleukemia.1 Clonal chromosomal abnormalities are fre-quently identified in MDS patients and may criticallyaffect the malignant transformation.2,3 Among the mostcommon cytogenetic abnormalities is the interstitialdeletion of the long arm of chromosome 5 [del(5q)]identified in 16% to 28% of MDS patients.4,5 Recently,it was shown that the immunomodulating drug (IMiD)lenalidomide, a 4-amino-glutarimide analogue ofthalidomide, may substantially improve or even restoreerythropoiesis in MDS patients with del(5q) by sup-pressing the abnormal clone.6,7 Based on the profoundhematologic and cytogenetic responses in the lower riskpatients, lenalidomide was approved for the treatmentof the transfusion-dependent low/intermediate-1 riskMDS patients, according to the International PrognosticScoring System (IPSS),8 displaying del(5q) alone or withadditional chromosomal abnormalities.9

The precise mechanism of action of lenalidomide inMDS and its biologic effects on the BM hematopoieticand microenvironmental cells remain largely unknown.In vitro studies have shown that lenalidomide displaysa direct inhibitory effect selectively on the hematopoi-etic progenitor cells of the del(5q) clone, but does notaffect the growth of the cytogenetically normal cells inMDS patients.10 Interestingly, a pro-proliferative anderythropoiesis-promoting effect of lenalidomide on nor-mal BM hematopoietic progenitor cells has been report-ed.11,12 In association to the direct effects, lenalidomidemay indirectly affect the survival and growth ofhematopoietic progenitor cells in MDS through itsimmune-modulating, anti-angiogenetic and adhesion-modulating properties.13,14 In vitro studies have shownthat lenalidomide down-regulates the production of thepro-inflammatory cytokines tumor necrosis factor !(TNF-!), interleukin-1 " (IL-1"), and transforminggrowth factor "-1 (TGF-"1) by activated monocyteswhile up-regulates IL-2 and interferon-gamma (IFN-#)production promoting the activation of T- and naturalkiller (NK)-cells.15;16 A co-stimulatory effect of lenalido-mide on T-cell responses following T-cell receptor acti-vation as well as an inhibitory effect on T-regulatorycells have been also reported.14,17 Lenalidomide, likeother IMiDs, may inhibit the secretion of angiogeneticcytokines by both BM hematopoietic and microenvi-ronment cells and may also alter a broad range ofresponses induced by angiogenetic and cell adhesionmolecules.18,19

A number of elegant clinical studies have substantiat-ed the exciting effect of lenalidomide on erythropoiesisof MDS patients with del(5q) and have resolved clinical-ly relevant practical considerations of the treatment.20-22

The effect of the lenalidomide therapy, however, on thereserves, functional and survival characteristics of BMhematopoietic cells and the function of BM stromal cells

has not been extensively studied. In the current studywe have globally examined the BM hematopoiesis inassociation with the clinical response in a number oflower risk MDS patients with del(5q) followinglenalidomide therapy. We have specifically evaluated,prior and post treatment, the number and clonogenicpotential of the BM erythroid, myeloid and megakary-ocytic progenitor cells, the apoptotic characteristics andadhesion molecule expression of CD34+ cells as well asthe hematopoiesis supporting capacity and the pro-inflammatory cytokine, angiogenetic and adhesion mol-ecule production by BM stromal cells. Changes in thenumber and activation status of PB lymphocyte subsetshave been also evaluated.

Design and Methods

PatientsTen white patients with de novo MDS according to

French-American-British (FAB) criteria, eight femalesand two males, aged 60 to 80 years (median age 71years), were enrolled in the study. All patients haddel(5q) as isolated cytogenetic abnormality, they hadlow or intermediate-1 risk according to the IPSS andwere transfusion-dependent requiring at least 2 units ofred cells during the last 8 weeks before enrollment.8The patients were assigned to receive lenalidomide(Caps Revlimid; Genesis Pharma, Greece) at the stan-dard dose of 10 mg/day for 21 days of every 28-daycycle without any additional treatment, except for redcell and platelet transfusion when required. Baseline(week 0) and following treatment (week 24) evaluationincluded PB cell counts and flow-cytometric analysis,BM aspirate and biopsy for morphologic, flow-cytomet-ric and cytogenetic analysis and in vitro studies ofhematopoiesis. PB counts, differential and basic serumchemistry survey were monitored weekly. Assessmentof clinical response was based on previously reportedcriteria.8 BM samples were also obtained from 30 hema-tologically normal subjects, age- and sex-matched to thepatients, whereas 10 more healthy controls were usedfor the CD34+ cell sorting for the recharging experi-ments. Informed consent according to the HelsinkiProtocol was obtained from all subjects and the studyhas been approved by the Institutional EthicsCommittee and the Hellenic Drug Organization.Detailed patient characteristics are depicted in Table 1.

Bone marrow samplesBM samples from posterior iliac crest aspirates were

diluted 1:1 in Iscove’s modified Dulbecco’s medium(IMDM; GibcoBRL, Life Technologies, Palsley,Scotland), supplemented with 100 IU/mL penicillin-streptomycin (PS; Gibco) and 10 IU/mL preservative-free heparin (Sigma, St Louis, MO, USA). Diluted BMsamples were centrifuged on Lymphoprep (NycomedPharma AS, Oslo, Norway) to obtain the mononuclearcells (BMMCs).

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Purification of CD34+ cellsCD34+ cells were isolated from BMMCs by indirect

magnetic labelling (magnetic activated cell sorting;MACS isolation kit, Miltenyi Biotec GmbH, Germany)according to the manufacturer’s protocol. In each exper-iment, purity of CD34+ cells was greater than 96% asestimated by flow-cytometry.

Reserves of BM progenitor and precursor cells Flow-cytometry was used to quantitate the BM

CD34+ progenitor cells and their subpopulations.Specifically, 1$106 BMMCs were triple-stained withphycoerythrin (PE)-conjugated mouse anti-humanCD34 mAb (581; Beckman-Coulter, Marseille France),phycoerythrin-cyanine5 (PC5)-conjugated CD45 (J.33)and fluorescein isothiocyanate (FITC)-conjugated CD33(D3HL60.251) or CD71 (YDJ1.2.2) or CD61 (SZ21)mAbs (Beckman-Coulter) for the estimation of themyeloid, erythroid, and megakaryocytic progenitorcells, respectively. CD45/CD34 stained BMMCs werealso stained with fluorescence-conjugated mAbs (allpurchased from Beckman-Coulter unless otherwiseindicated) against the adhesion molecules CD11_ (25.3),CD44 (MEM.85; Caltag Laboratories, Burlingame, CA,USA), CD48 (J4.57), CD54 (84H10), CD49d (HP2/1),CD49e (SAM1), CD62L (DREG56) and the chemokinereceptor of stromal derived factor-1 (SDF-1) CXCR4(CD184; 12G5). PE-, FITC- and PC5-conjugated mouseIgG of appropriate isotype served as negative controls.Data were acquired and processed on 500,000 eventsusing an Epics Elite model flow-cytometer (Coulter,Miami, FL, USA). Analysis was performed in the gate ofcells with low forward scatter (FSC) and low side scat-ter (SSC) properties gated on the CD45 positive cellsaccording to the CD45PC5/SSC scattergram. For thequantification of the erythroid precursor cells, 1$106

BMMCs were stained with anti-Glycophorin A(GlycoA)-PE (11E4B7.6) and anti-CD36-FITC (FA6-152)mAbs (Beckman-Coulter) or with PE- and FITC-conju-gated mouse IgG to identify the CD36+/GlycoA+ andCD36–/GlycoA+ early and mature erythroid precursorcells, respectively.23,24

Study of apoptosis Flow-cytometry and 7-amino-actinomycin D staining

(7AAD) was used to study apoptosis in the BM cell sub-populations. For the study of apoptosis in the CD34+

cell fraction, aliquots of 1$106 BMMCs stained withanti-CD34-PE mAb as above, were further stained with100 mL 7AAD (200 mg/mL) (7AAD; Calbiochem-Novabiochem, La Jolla, CA, USA) as previouslydescribed.25,26 For the analysis, a scattergram was createdby combining SSC with CD34 fluorescence in the gateof cells with low FSC and SSC properties and a secondscattergram was created by combining FSC with 7AADfluorescence to quantitate 7AAD-negative (live), -dim(early apoptotic) and –bright (late apoptotic/dead) cellsin the gate of CD34+ cells (Figure 1). For the study ofapoptosis in the erythroid cell subpopulations, aliquotsof cells stained with the combination anti-CD34-PE/anti-CD71-FITC or anti-GlycoA-PE/anti-CD36-FITC were further stained with 7AAD. Quantificationof the live, early and late apoptotic cells was performedin the gates of CD34+/CD71+, CD36+/GlycoA+, andCD36–/GlycoA+ erythroid cell populations as previous-ly detailed24,27 (Figure 1) .

Clonogenic assays Erythroid and myeloid colony-forming units: For the

estimation of the erythroid and myeloid colony-form-ing units, we cultured 105 BMMCs in 35-mm Petri dish-es in 1 mL methylcellulose culture medium (Stem CellTechnologies, Vancouver, BC, Canada) supplementedwith 5 ng/mL granulocyte-macrophage colony stimulat-ing factor (GM-CSF; R&D Systems, Minneapolis, MN),50 ng/mL IL-3 (R&D Systems) and 2 IU/mL Epo(Janssen-Ciliag, Athens, Greece) as previouslydetailed.28 Following 14 days of culture at 370C-5%CO2 fully humidified atmosphere, colonies werescored and classified as BFU-E (erythroid-burst formingunits) and total CFU-GM (granulocyte- plusmacrophage- plus granulocyte-macrophage colonyforming units). The total sum of colonies was character-ized as colony-forming cells (CFCs).

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Table 1. Clinical and laboratory data of the patients studied.*UPN Age/Sex Duration FAB IPSS BM Karyotype BM Blasts Hgb Neutro Plts Transfusion

(months) (%) (g/dL) (x109/L) (x109/L) dependence#

1 81/F 31 RA Low 46__,del(5)(q13q33)[20/20] 2 10.5 2.1 135 62 62/M 14 RAEB INT-I 46__,del(5)(q13q33)[20/20] 10 5.7 4.5 120 353 67/F 14 RA Low 46XX[2/20], 46XX, del(5)(q13q33)[18/20] 1 9.7 2.4 196 64 77/M 84 RA INT-I 46XY,del(5)(q13q33)[20/20] 3 7.2 5.3 197 225 78/F 48 RA INT-1 46XX,del(5)(q13q33)[20/20] 4 8.5 1.0 498 186 59/F 2 RA INT-1 46__,del(5)(q13q33) [20/20] 10 7.4 1.8 150 67 79/F 4 RA Low 46__,del(5)(q13q33)[20/20] 4 8.1 2.5 272 68 74/F 46 RA Low 46XX,del(5)(q13;q33)(20/20) 1 7 4.5 213 249 64/F 17 RAEB INT-I 46__,del(5)(q13;q33) (10/20), 46XX (10/20) 9 7.3 0.5 23 5110 80/M 11 RA Low 46__,del(5)(q13;q33) (12/20), 46XX(8/20) 2 7.5 3.0 259 7UPN: unique patient number; FAB: French-American-British classification; IPSS: International Prognostic Scoring System; BM: bone marrow; Hgb: hemoglobin;Neutro: neutrophils; Plts: platelets. * At the day of BM aspiration,before lenalidomide administration. #Units of red blood cells required during the last 6 months.

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Megakaryocye colony-forming unitsFor the quantification of the megakaryocyte colony-

forming units (CFU-Meg), we cultured 5$105 BMMCsper chamber of a double-chamber slide using a commer-cially available culture medium (MegaCult-C, StemCellTechnologies), according to the manufacturer’s instruc-tions. Following 10 to 12 days incubation at 37°C-5%CO2 humidified atmosphere, colonies were scoredafter fixation and staining of culture slides with anti-CD41 mAb (5B12; Dako, Glostrup, Denmark) using thealkaline phosphatase anti-alkaline phosphatase tech-nique (APAAP), as previously described.29 Results wereexpressed as total CFU-Meg colonies (pure CFU-Meg +Mixed CFU-Meg).

Assessment of BM stromal cell function Standard LTBMCs. 107 BMMCs were grown accord-

ing to the standard technique28 in 10 mL IMDM sup-plemented with 10% fetal bovine serum (FBS; Gibco),10% horse serum (Gibco), 100 IU/mL PS, 2 mmol L-glu-tamine and 10-6 mol hydrocortisone sodium succinate(Sigma) and incubated at a 330C-5%CO2 humidifiedatmosphere. At weekly intervals, cultures were fed bydemi-depopulation and nonadherent cells (NACs) werecounted and assayed for CFCs as above.

Cytokine measurement in LTBMC supernatants. Cell-free supernatants of confluent LTBMCs (week 3-4)before and after lenalidomide treatment were stored at–70°C for quantification of TNF-!, TGF-"1, SDF-1a,fibroblast growth factor (FGF), vascular endothelialgrowth factor (VEGF), intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)and E-Selectin using an enzyme-linked immunosorbentassay (ELISA). All ELISA kits were purchased from R&DSystems except for the TNF-a kit (BiosourceInternational Inc, California, USA).

Recharged LTBMCsTo test the hematopoiesis supporting capacity of

patient LTBMC stromal cells independently of theautologous cells, we used a 2-stage culture procedure as

previously described.30,31 In brief, confluent LTBMC stro-mal layers from patients and normal controls were irra-diated (10 Gy) and recharged with 5x104 allogeneic nor-mal CD34+ BM cells. In each experiment, flasks wererecharged in triplicate and CD34+ cells from the samenormal control were used to test patient and normal cul-tures. Cultures were monitored weekly by determiningthe number of CFCs in the NAC fraction.

PB lymphocyte subsetsTwo-color flow-cytometry was used for the analysis

of PB lymphocyte subsets. In brief, 100 mL aliquots ofEDTA-anticoagulated PB samples were stained asdescribed above with a combination of PE- or FITC-conjugated mAbs (purchased from Beckman-Coulterunless otherwise indicated). In particular, anti-CD3(UCHT1) was combined with each of the followingmAbs representing T-cell activation markers: anti-CD25(IL-2 receptor; B1.49.9), anti-CD95 (anti-Fas) (LOB3/17;Serotec, Oxford, United Kingdom), anti-CD38 (T16),anti-CD69 (TP1.55.3) and anti-CD71. The proportion ofB- and NK-cells was also estimated using the anti-CD19(J4.119) and anti-CD16 (3G8), anti-CD56 (N901), anti-CD57 (NC1) mAbs, respectively. Red blood cell lysisand cell fixation was performed using the Q-prepreagent system (Coulter, Luton, England). The datawere acquired as above and analysis was performed on10,000 events in the gate of cells with low FSC and lowSSC properties where lymphocytes are included.Results were expressed as proportions of cells express-ing each mAb. Furthermore, by dividing the proportionsof double positive cells using the above described mAbcombinations by the percentages of total CD3+ cells, weestimated the proportions of activated cells within theT-lymphocyte population.32

Statistical analysis Data were analyzed in the GraphPAd Prism statistical

PC program (GraphPad Software, San Diego, CA). TheStudent’s t-test for paired samples was used to definedifferences before and after treatment with lenalido-

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Figure 1. Flow-cytometric analysis of BMcells. Scattergram (A) of forward scatter(FSC) versus side scatter (SSC) to allowgating on cells with low FSC and low SSCproperties (R1). Scattergram (B) of anti-CD34 fluorescence versus SSC gated onR1, to allow gating on CD34+ cells (R2).Scattergram (C) of FSC versus 7AAD fluo-rescence gated on the CD34+ cells (R2)showing the live (R3), early apoptotic (R4)and late apoptotic/dead (R5) cells.Apoptosis was similarly studied within theCD34+/CD71+ erythroid progenitor cells(R6) (plot D) and the CD36+/GlycoA+ (R7)and CD36–/GlycoA+ (R8) erythroid precur-sor cells (plot E). An example of apoptosisin the ungated cells is depicted in scatter-gram F to clearly show the live (R9), early(R10) and late apoptotic/dead (R11) cells.

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mide. Standard 2-way analysis of variance (2-wayANOVA) was applied to define differences in the num-ber of CFCs in LTBMCs either between the patientsprior and post treatment or between patients andhealthy controls. The Mann-Whitney test was used tocompare flow cytometry and colony data betweenpatients post therapy and healthy controls. Groupeddata are expressed as mean ± 1 standard deviation (SD).

Results

Reserves, survival and immunophenotypiccharacteristics of BM progenitor/precursor cells

Overall, the percentage of total CD34+ cells were sig-nificantly reduced following treatment (2.02%±2.13%)compared to baseline (11.33%±5.09%, p=0.0005) sug-gesting a decrease of blast cells in patients’ BM (Figure

2). A significant decrease was also obtained in the per-centage of the myeloid CD34+/CD33+ (0.13%±0.27%),megakaryocytic CD34+/CD61+ (0.45%±0.48%) and ery-throid CD34+/CD71+ (0.57%±0.65%) progenitor cellsfollowing treatment compared to baseline (1.02% ±1.15%, 5.20%±2.76%, 3.73%±4.54%, respectively)(p=0.0241, p=0.0003, p=0.0492, respectively). Thisdecrease probably reflects the reduction of the totalCD34+ cells after therapy and not a frank reduction ofthe myeloid, megakaryocytic and erythroid progenitorcells. Interestingly, the proportion of CD34+/CD61+ andCD34+/CD71+ cells of patients post therapy were simi-lar to those of the healthy subjects (0.50% ± 0.31% and0.59%±0.24%, respectively) (p=0.3907 and p=0.0796,respectively). Regarding the erythroid precursor cells, asignificant increase was obtained in the proportion ofthe CD36+/GlycoA+ and CD36-/GlycoA+ subpopula-tions post therapy (13.35%±8.90% and10.45%±15.18%, respectively) compared to baseline(4.04%±2.87% and 5.61%±9.59%, respectively)(p=0.0068 and p=0.0472, respectively) (Figure 2). Thesefindings indicate a lenalidomide-mediated inhibition ofthe del(5q) progenitor pool which display an inherentdefect in erythroid differentiation and a parallel expan-sion of the non-del(5q) progenitors which show bettererythroid differentiation potential. Interestingly, how-ever, the proportion of the more mature CD36-/GlycoA+ cells, remained lower than the healthy con-trols (33.21%±5.56%) (p=0.0017).

The survival characteristics of patient BM progenitorand precursor cells before and after therapy are also pre-sented in Figure 2. The proportion of apoptotic(7AADdim) cells within the CD34+ cell fractionincreased significantly following lenalidomide therapy(16.03%±11.99%) compared to pre-treatment values(2.90%±2.56%; p=0.0070). Similarly, a significantincrease was obtained post-therapy in the proportion ofapoptotic cells within the CD36+/GlycoA+

(25.27%±16.33%) and CD36-/GlycoA+ (8.76%±9.01%)early and mature erythroid precursor cells compared tobaseline (7.19%±7.58% and 2.87%±3.29%, respective-ly) (p=0.0033 and p=0.0315, respectively). However, theproportion of apoptotic cells within the CD34+,CD36+/GlycoA+ and CD36-/GlycoA+ cell compartments

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Table 2. Flow-cytometric analysis of adhesion molecule expressionon BM CD34+ cells.% of cells expressing Pre-Lenalidomide Post-Lenalidomide p the antigen within (n=10) (n=10) value*the CD34+ cell fraction

CD11a 38.55±10.55# 66.46±20.74 0.0034median (range) 39.63 (22.11-54.46) 65.34 (38.89-100)CD44 83.31±17.80 87.15±14.10 0.6312median (range) 88.80 (39.32-98.36) 87.32 (56.89-100)CD48 10.82±9.07 48.73±35.05 0.0150median (range) 7.30 (0.79-26.61) 48.52 (5.00-100)CD49d 39.69±19.31 62.57±14.48 0.0037median (range) 38.96 (14.15-66.14) 65.35 (37.50-81.81)CD49e 18.54±13.39 7.72±11.02 0.0982median (range) 16.47 (0.97-38.65) 3.16 (0.20-33.33)CD54 8.81±5.89 28.41±17.28 0.0093median (range) 8.27 (0.10-19.58) 25.31 (4.30-58.33)CD62L 21.72±18.29 21.06±17.97 0.9392median (range) 19.76 (0-55.96) 16.61 (0-50.00)CXCR4 2.96±1.96 18.51±17.22 0.0159median (range) 3.23 (0.82-5.60) 14.91 (2.43-50.00)* Statistical analysis was performed using the Student’s t-test for paired samples.# Values are expressed as means ± SD. The median and range values are alsoshown.

Figure 2. Flow-cytometric evaluation ofthe reserves and survival characteristicsof BM progenitor and erythroid precursorcells before and after lenalidomide thera-py. The left bars represent the mean pro-portion (± SEM) of BM CD34+ progenitorand CD36+/GlycoA+ and CD36-/GlycoA+

erythroid precursor cells before and aftertreatment. The right bars represent themean proportion (± SEM) of apoptotic(7AADdim) cells within the CD34+,CD36+/GlycoA+ and CD36-/GlycoA+ cellcompartments before and after therapy.Comparison was performed using theStudent’s t-test for paired samples and Pvalues are indicated. SEM: Standard Errorof the Mean.

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remained higher in patients compared to healthy con-trols (4.91%±4.63%, 5.04%±3.04% and 2.46%±2.43%,respectively) (p=0.0092, p=0.041, and p=0.0378 respec-tively).

Changes in the expression of adhesion molecules inthe CD34+ cell fraction before and after therapy are pre-sented in Table 2. A statistically significant increase wasobtained in the proportion of CD34+ cells expressing theCD11a, CD49d and CD54 antigens following therapycompared to baseline (p=0.0034, p=0.0037 andp=0.0093, respectively) suggesting that lenalidomideimproves the adhesion capacity of BM hematopoieticprogenitor cells in the BM microenvironment structures.In favor of this hypothesis was the increased proportionof CD34+ cells expressing the CXCR4 chemokine recep-tor following therapy compared to baseline (p=0.0159).No statistically significant difference was obtained inthe proportion of CD34+ cells expressing the CD44,CD49a, and CD62L adhesion molecules following ther-apy. Interestingly, a statistically significant increase wasobtained in the proportion of CD48+ cells within theCD34+ cell fraction following therapy compared tobaseline (p=0.0037) possibly indicating the increase inthe committed progenitor cell number.33,34

Clonogenic progenitor cells The number of clonogenic progenitor cell pre- and

post-lenalidomide therapy are depicted in Figure 3A.The frequency of CFCs obtained by BMMCs increasedsignificantly following therapy (2060±1652 per 107BMMCs) compared to baseline (420±655 per 107BMMCs; p=0.0074). This increase was due to theimprovement of both CFU-GM and BFU-E post-therapy(1670±1497 and 390±292 per 107 BMMCs, respectively)compared to pre-treatment (340±481 and 80±187 per107 BMMCs, respectively) (p=0.0159 and p=0.0029,respectively). A significant increase was also obtained inthe number of CFU-Meg post-therapy (1988±589 per

107 BMMCs) compared to baseline (740±500 per 107BMMCs; p=0.0002). These data suggest an improve-ment of the clonogenic potential of patient BMMCs fol-lowing therapy. However, the number of the clonogenicprogenitor cells, namely the CFU-GM, BFU-E and CFU-Meg remained lower in patients post therapy comparedto healthy controls (5017±2634, 3400 ± 1580 and 2940± 1097 per 107 BMMCs, respectively) (p=0.0019,p<0.0001 and p=0.0193 respectively) suggesting thepossible persistence of clonal cells in the hematopoieticprogenitor cell pool.

BM stromal cell function Typical confluent stromal layers, consisting of cells of

hematopoietic and mesenchymal origin that mimic theBM microenvironment,35 were formed over the first 3-4 weeks in patient LTBMCs prior- and post-therapy.The number of CFCs in the NAC fraction increased sig-nificantly following lenalidomide therapy compared tobaseline (F=14.338, p<0.001), over a period of 5 weeksof culture (Figure 3A). This increase probably reflectsthe improvement of the clonogenic potential of BM pro-genitor cells post-therapy. Alternatively, this increasemay also indicate an improvement in the hematopoiesissupporting capacity of LTBMC adherent cells after treat-ment. To investigate this hypothesis, we evaluated thecapacity of irradiated LTBMC stromal layers from thepatients to support the growth of normal CD34+ cellspre- and post-therapy. Before lenalidomide therapy, theCFC recovery from NACs was significantly lower in thepatients compared to healthy controls (F=11.014,p<0.01) suggesting a defective hematopoiesis support-ing capacity of patient stromal cells. Following therapy,however, the number of CFCs in the NAC fraction didnot differ significantly between patients and healthycontrols (F=0.016, p>0.05), suggesting a substantialimprovement in the capacity of patient stromal layers tosupport hematopoiesis. To probe further the mecha-

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| 6 | haematologica | 2009; 94(12)

Figure 3. BM clonogenic progenitor cellsand PB lymphocyte subsets before andafter lenalidomide therapy. The bars inthe upper graph A depict the mean num-ber (±SEM) of CFU-Meg, CFU-GM and BFU-E colonies obtained by 107 BMMCs andthe bars in the lower graph A the meancolony forming cell (CFC) numbers(±SEM) in long-term BM culture super-natants over 5 weeks of culture, beforeand after therapy. Analysis has been per-formed by means of Student t-test forpaired samples (upper graph) and 2-wayANOVA (lower graph) and the P and F val-ues are indicated. The bars in graph Brepresent the mean proportion (±SEM) ofPB lymphocyte subpopulations (upperpanel) and the mean percentage of cellsexpressing markers of activation withinthe CD3+ cell fraction (lower panel),before and after therapy. Analysis wasperformed by means of Student t-test forpaired samples and P values are shown.SEM; Standard Error of the Mean.

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nism underlying this improvement, we evaluated thelevels of hematopoiesis-related cytokines in LTBMCsupernatants reflecting essentially the cytokine produc-tion in patients’ BM microenvironment. A significantincrease was observed in the levels of SDF-1a andICAM-1 post-therapy compared to baseline (p=0.0297and p=0.0059, respectively) whereas no statistically sig-nificant difference was observed in the levels of TNF-a,TGF-"1, FGF, VEGF, VCAM-1 and E-Selectin (Table 3).

PB lymphocyte subsetsActivated T-lymphocytes have been implicated in the

pathophysiology of MDS.36 We have therefore studiedpatient PB lymphocyte subsets before and afterlenalidomide therapy, focusing mainly on the expres-sion of markers of T-cell activation. We found that theproportion of PB CD3+ cells increased significantly fol-lowing therapy (74.54%±6.06%) compared to baseline(65.83%±6.15%; p=0.006) whereas the proportion ofCD19+, CD16+ , CD56+ and CD57+ cells remainedunchanged (Figure 3B). A significant increase wasobserved in the proportion of activated T-cells aftertreatment as was indicated by the increased proportionof CD69+, CD38+, CD25+, CD95+ and CD71+ cells with-in the CD3+ cell fraction (7.86%±3.36%,22.16%±8.01%, 8.04%±3.53%, 29.36%±12.62%,3.70%±1.82%, respectively) compared to baseline(3.71%±2.17%, 13.76%±8.97%, 6.01%±3.11%,14.96%±9.15%, 2.03%±0.99%, respectively)(p=0.0253, p= 0.0274, p=0.0070, p=0.0162, p=0.0093,respectively) (Figure 3B).

Response evaluationHematologic and cytogenetic responses were

assessed according to the modified criteria of theInternational Working Group.37 Eight patients displayeda major erythroid response as was demonstrated by thetransfusion independence, whereas two patients dis-played a minor response with 59% and 55% reductionin transfusions, respectively. In all cases improvementwas sustained for at least eight consecutive weeks.Hematologic improvement was associated with the pat-tern of cytogenetic responses. Specifically, patients withmajor erythroid improvement displayed also majorcytogenetic response as was demonstrated by theabsence of the del(5q) abnormality on the standardmetaphase analysis. The two patients with the minorerythroid improvement displayed a minor cytogeneticresponse as was shown by the reduction in the numberof abnormal cells in metaphases at a percentage of 52%and 95%, respectively.

Discussion

The IMiD lenalidomide has profound therapeuticeffects in MDS patients with del(5q).9 Several cellularactivities for lenalidomide have been described includ-ing anti-angiogenic and anti-inflammatory actionsthrough regulation of cytokine production and modula-tion of T-, NKT-, T-regulatory and NK-cell functions.13-

19,,38 Although lenalidomide seems to mainly target theBM microenvironment structures, a direct effect of thedrug on the clonal hematopoietic cells has beendescribed through inhibition of proteins critical for cellsurvival or stimulation of tumor suppressor genes onthe 5q region.10,39 It seems, however, that many mecha-nisms of action of lenalidomide on patients’hematopoiesis remain still undefined. Studies investi-gating the mode of action of lenalidomide in MDSpatients with del(5q) have been mainly based on the invitro and/or ex vivo incubation of hematopoietic cellswith the drug and examination of changes at cellularand molecular level. In our study, we have investigatedex vivo the effect of lenalidomide treatment on thereserves and functional characteristics of BM progeni-tor/precursor and microenvironmental cells inlow/intermediate-I risk MDS patients with del(5q), inassociation to the clinical effect. In accordance with pre-viously reported data,7 80% of our patients becametransfusion-independent whereas 20% of the patientsdisplayed a minor erythroid response with significantreduction of transfusion requirements. Patients withmajor erythroid improvement displayed also majorcytogenetic response with disappearance of the del(5q)abnormality whereas patients with minor erythroidimprovement displayed also a minor cytogeneticresponse, suggesting that cytogenetic and hematologicpatterns of response correlate significantly.

The study of the reserves and functional characteris-tics of BM progenitor cells showed that the proportionof CD34+ cells decreased significantly following therapyand this decrease was associated with a significant

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Table 3. Cytokine levels in LTBMC supernatants.Cytokine Pre-Lenalidomide Post-Lenalidomide p

(n=10) (n=10) value*

SDF-1a (pg/mL) 277.81±153.40# 782.52±665.35 0.0297median (range) 250.77 493.23

(125.83-488.50) (194.03-1891.49)FGF (pg/mL) 2.91±1.45 2.75±1.91 0.8509median (range) 2.49 (1.32-5.77) 1.90 (1.55-7.88)VEGF (pg/mL) 1642.09±1207.48 1502.41±1041.76 0.5748median (range) 1877.26 1536.56

(131.95-3404.90) (123.38-3493.01)TNF-" (pg/mL) 1.14 ± 0.58 1.22±0.56 0.6241median (range) 1.09 (0.57-2.02) 1.20 (0.57-2.10)VCAM (ng/mL) 70.98±4.20 87.00±29.29 0.1359median (range) 71.58 (65.57-77.57) 80.57 (65.57-166.02)E-Selectin (ng/mL) 0.17±0.15 0.09 ± 0.11 0.2146median (range) 0.14 (0.05-0.50) 0.05 (0.05-0.41)ICAM-1 (ng/mL) 1.62±0.57 2.67±0.75 0.0059median (range) 1.65 (0.92-2.67) 2.96 (0.92-3.25)TGF-"1(pg/mL) 474.83±283.27 440.97±331.69 0.3887median (range) 455.84 323.64

(48.57-938.56) (26.99-931.09)*Statistical analysis was performed using the Student’s t-test for paired samples.# Values are expressed as means ± SD.The median and range values are alsoshown.SDF: stromal Derived Factor; FGF: fibroblast Growth Factor;VEGF: vascularEndothelial Growth Factor;TNF: tumor necrosis factor; VCAM: vascular CellAdhesion Molecule; ICAM, Intercellular Adhesion Molecule;TGF: transformingGrowth Factor

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increase in the proportion of apoptotic cells within thiscell compartment. Although a discrimination betweenclonal and normal CD34+ cells has not been performed,it seems reasonable to accept that the CD34+ cellsbefore therapy belong mainly to the malignant cloneand accordingly, display apoptosis resistance and sur-vival advantage over the apparently normal CD34+ cellspost therapy.40 In addition, we might hypothesize thatthe lenalidomide-induced SPARC up-regulation, whichhas been shown in both clonal and normal cells,10 mighthave a role. Specifically, it has been shown that SPARC,in addition to its anti-proliferative, anti-adhesion andanti-angiogenetic function may display an apoptosis-inducing effect.41,42 In keeping with the increased propor-tion of apoptotic CD34+ cells post therapy was theaccelerated apoptosis of the erythroid progenitor/pre-cursor cells following treatment indicating probably therecovery of the normal, non-clonal cells. In favor of thishypothesis was the improvement in the number andthe clonogenic potential of BM erythroid cells post ther-apy as was demonstrated by the proportion of GlycoA+

cells and the frequency of BFU-E in the BMMC fractionbut also by the hematologic improvement.

In association to the increased BFU-E colony num-bers, we observed a significant increase in the CFU-GMand CFU-Meg colony recovery by BMMCs post thera-py, indicating a global beneficial effect of lenalidomideon the reserves and clonogenic potential of BM progen-itor cells. Previous studies have also shown significantchanges in the multipotent and BFU-E colony numbersin MDS patients responding to lenalidomide therapycompared to non-responders.7 However, a beneficialeffect of the drug on the clonogenic potential of CFU-Meg progenitor cells has not been reported so far. Thispositive action of lenalidomide on the clonogenic poten-tial of BM progenitor cells is in agreement with previousobservations suggesting a beneficial effect of the drugon the expansion and proliferation rate of normalCD34+ cells.11

It has been hypothesized that lenalidomide may indi-rectly affect the functional characteristics of BMhematopoietic cells in MDS patients with del(5q) byaltering the structures, the cellular components and thehumoral compounds of the BM microenvironment.Specifically, it has been shown that treatment withlenalidomide decreases the BM microvessel densityindicating an anti-angiogenic activity which has beenassociated with a reduction in the levels of pro-angio-genic cytokines7,10,43 It has been also shown that lenalido-mide may decrease the production of pro-inflammatorymediators in the BM microenvironment and may altercell-to-cell interactions through modification of expres-sion of adhesion molecules and stimulation of respons-es of cytotoxic T- and NK-cells.39 To probe the effect oflenalidomide on the hematopoiesis supporting potentialof BM stroma, we used the LTBMC system which haslong been considered as a representative in vitro modelmimicking the BM microenvironment.35 We observed asubstantial improvement in the capacity of patientLTBMC adherent layers to sustain the autologous andnormal hematopoietic progenitor cell growth followinglenalidomide treatment compared to baseline. To gain

insight into the mechanisms underlying the beneficialeffect of lenalidomide on the BM stromal cell function,we evaluated the levels of soluble adhesion molecules,pro-inflammatory and pro-angiogenic cytokines inLTBMC supernatants. In accordance with previouslyreported data showing minimal changes in VEGF andFGF levels in BM plasma following lenalidomide thera-py,7 we also found insignificant alterations in LTBMCsupernatant levels of these molecules after treatment.Minor changes were also observed in the levels of thecytokines TNF-! and TGF-"1 and the soluble adhesionmolecules VCAM-1 and E-selectin following lenalido-mide treatment. However, a profound increase wasobtained in the levels of supernatant SDF-1a and ICAM-1 post therapy that was associated with a significantincrease in the expression of the respective membraneligands CXCR4 and CD11a on CD34+ cells. These datasuggest that lenalidomide favors the maintenance ofCD34+ in the BM by inducing the CXCR4/SDF-1a andCD11a/ICAM-1 interactions between the hematopoiet-ic and stromal cells. The increased expression of ICAM-1 (CD54) and CD49d on CD34+ cells post therapy cor-roborates this assumption. Furthermore, it has beenshown that the del(5q) early hematopoietic stem cellsare unable to repopulate nonobese diabetic/severe com-bined immunodeficiency mice in standard transplanta-tion models suggesting a defect in cell homing thatmight be associated with a defective SDF-1a/CXCR4axis.44 Based on these studies suggesting that the earlyhematopoietic stem cell compartment in patients withdel(5q) is dominated by the clonal cells with possibledefective homing properties,44 we may hypothesize thatthe increase in the CXCR4 expression in patients posttherapy may reflect a lelalidomide-induced inhibition ofthe abnormal clone and expansion of the normal clonewith normal homing properties. A significant increasewas observed in the expression of CD48 on CD34+ cellspost therapy compared to baseline. This antigenbelongs to the SLAM family stem/progenitor cell sur-face receptors and it is mainly expressed on the commit-ted progenitors rather than on primitive stem cells.33;34Accordingly, the increased expression of CD48 withinthe CD34+ cell population following therapy may sim-ply reflect the increased number of clonogenic progeni-tor cells obtained by lenalidomide treatment that nor-mally express CD48. However, CD48 represents also aco-stimulatory receptor for CD2 and 2B4 moleculesnormally expressed on T- and NK cells45 and its ligationhas been reported to prolong cellular interactions and tofacilitate the T- and NK-mediated signaling.46,47

Therefore, this increased expression of CD48 on patientCD34+ cells may indicate a lenalidomide-inducibleeffect that may contribute to the hematopoietic progen-itor cell apoptosis through T- and/or NK-cell mediatedeffects. Finally, we found a significant increase in thepercentage of T-cells following lenalidomide therapythat was associated with an activation profile as wasdemonstrated by the increased proportion of T-cellsexpressing the CD69, CD38, CD25, CD95, and CD71markers of activation.

In conclusion, the data of this study provide newaspects of the mechanism of action of lenalidomide in

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patients with lower risk MDS and del(5q) while alsoconfirm the beneficial effect of lenalidomide in theinduction of erythroid and cytogenetic responses.According to our data, the clinical effect of lenalidomideis associated with a significant increase in the number oferythroid, myeloid and megakaryocytic colony-formingcells and substantial improvement in the hematopoiesissupporting capacity of BM stroma. Lenalidomideinduces significant alterations in the adhesion profile ofhematopoietic progenitor cells such as overexpressionof membrane CXCR4, CD54, CD11a and CD49d andoverproduction of SDF-1a and ICAM-1 in the BMmicroenvironment. The induction of the SLAM antigenCD48 on patient CD34+ cells by lenalidomide may beassociated with its apoptosis-inducing effect throughco-stimulatory interactions between the CD34+ cells

and cytototoxic lymphocytes in the BM microenviron-ment.

Authorship and Disclosures

MX, MK, AP, KG, MP, PK, CS and CP performed lab-oratory work and contributed to the analysis and inter-pretation of data. AG, AS, VP, ZK, DL, EH, SK recruitedthe patients and contributed to the conception of thestudy. HAP was the principal investigator, designed andsupervised the research, analyzed the data, wrote thepaper and takes the primary responsibility for the paper.

The authors reported no potential conflicts of interest.

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