Cytometry Part B (Clinical Cytometry) 78B:154–168 (2010)

Bone Marrow Cells from MyelodysplasticSyndromes Show Altered ImmunophenotypicProfiles That May Contribute to the Diagnosisand Prognostic Stratification of the Disease:

a Pilot Study on A Series of 56 Patients

Sergio Matarraz,1 Antonio Lopez,1 Susana Barrena,1 Carlos Fernandez,1

Evan Jensen,1 Juan Flores-Montero,1 Ana Rasillo,1 Jose Marıa Sayagues,1

Maria Luz Sanchez,1 Paloma Barcena,1 Jesus Marıa Hernandez-Rivas,2

Carlos Salvador,3 Nuria Fernandez-Mosteirın,3 Manuel Giralt,3 Luis Perdiguer,4

Paula Laranjeira,5 Artur Paiva,5 and Alberto Orfao1*1Centro de Investigacion del Cancer (Instituto de Biologıa Molecular y Celular del Cancer; CSIC-USAL),

Servicio General de Citometrıa and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain2Servicio de Hematologıa, Hospital Universitario de Salamanca, Salamanca, Spain

3Servicio de Hematologia. Hospital Miguel Servet, Zaragoza, Spain4Servicio de Hematologia, Hospital de Alcaniz, Teruel, Spain

5Unidade de Citometria, Centro de Histocompatibilidade do Centro-Coimbra, Coimbra, Portugal

A heterogeneous spectrum of immunophenotypic abnormalities have been reported in myelodysplasticsyndromes (MDS). However, most studies are restricted to the analysis of CD341 cells and/or othermajor subsets of CD342 cells, frequently not exploring the diagnostic and prognostic impact ofimmunophenotyping.

Methods: We propose for the first time an immunophenotypic score (IS) based on the altered distribu-tion and immunophenotypic features of maturing/mature compartments of bone marrow (BM) hematopoi-etic cells in 56 patients with MDS that could contribute to a refined diagnosis and prognostic evaluationof the disease.

Results: Although MDS-associated phenotypes were detected in reactive BM, the overall immunopheno-typic profile of BM cells allowed an efficient discrimination between MDS and both normal and reactiveBM, once the number and degree of severity of the abnormalities detected per patient were simultane-ously considered in the proposed IS. Interestingly, increasingly higher IS were found among patients withMDS showing adverse prognostic factors and in low- versus high-grade cases. The most informative prog-nostic factors included the number of CD341 cells, presence of aberrant CD342/CD1171 precursors,decreased mature neutrophils and CD342 erythroid precursors, and increased numbers of CD362/lo ery-throid precursors; in addition, the IS was an independent prognostic factor for overall survival.

Conclusions: Assessment of immunophenotypic abnormalities of maturing/mature BM cells allows anefficient discrimination between MDS and both normal and reactive BM, once the number and degree ofseverity of the abnormalities detected are simultaneously scored. Interestingly, progressively higher ISwere found among patients with MDS with adverse prognostic features and shorter overall survival.VC 2010 Clinical Cytometry Society

Key terms: myelodysplastic syndromes; immunophenotypic score; survival

Grant sponsor: Instituto de Salud Carlos III, Ministerio de Sanidad yConsumo, Madrid, Spain; Grant number: RTICC RD06/0020/0035; Grantsponsor: Ministerio de Sanidad Consumo; Grant number: CP05/003 (toJ.M.S.); Grant sponsor: Fundacao para a Ciencia e a Tecnologia (FCT),Portugal; Grant sponsor: SFRH/BD/32097/2006 (to P.L.).

*Correspondence to: Alberto Orfao, Servicio General de Citometrıa.Centro de Investigacion del Cancer, Campus Miguel de Unamuno,37007-Salamanca, Spain. E-mail: orfao@usal.es.

Received 14 July 2009; Revision 27 January 2010; Accepted 28January 2010

Published online 2 March 2010 in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/cyto.b.20513

VC 2010 Clinical Cytometry Society

How to cite this article: Matarraz S, Lopez A, Barrena S, Fernandez C, Jensen E, Flores-Montero J, Rasillo A,Sayagues JM, Sanchez ML, Barcena P, Hernandez-Rivas JM, Salvador C, Fernandez-Mosteirın N, Giralt M, PerdiguerL, Laranjeira P, Paiva A, Orfao A. Bone marrow cells from myelodysplastic syndromes show altered immunophe-notypic profiles that may contribute to the diagnosis and prognostic stratification of the disease: a pilot studyon a series of 56 patients. Cytometry Part B 2010; 78B: 154–168.

Currently, diagnosis of myelodysplastic syndromes(MDS) mainly relies on morphologic features of bonemarrow (BM) precursors and maturing myeloid, ery-throid and megakaryocytic cells, once other potentialreasons for cytopenias and/or dysplasia have been ruledout (1–3). In addition to cytomorphology and histopa-thology, cytogenetic findings are also widely recognizedas being particularly valuable for (1) the diagnosis ofMDS; (2) the identification of specific subgroups of thedisease (e.g., 5q� syndrome) (3,4); and (3) prognosticstratification of patients at low versus high risk of devel-oping acute leukemia and/or dying (2,5–9).

In recent years, an increasingly high number of stud-ies has accumulated, which show occurrence of multi-ple and variable phenotypic abnormalities in MDS thatcould potentially contribute to the diagnosis and prog-nostic evaluation of the disease (10–15). Most frequentlyreported abnormalities include: increased numbers ofmyeloblasts, decreased granularity of maturing myeloidcells (11–13), asynchronous expression of maturation-associated markers, inappropriate expression of lymph-oid-related antigens (e.g., CD7), and either lack ordecreased reactivity for myeloid-associated antigens(e.g., CD13 and CD33), among other alterations (13). Inparallel, a number of reagent combinations have beenproposed that generate unique immunophenotypic pat-terns of antigen expression for both maturing neutro-phils and erythroid precursors, which are frequentlyaltered in MDS (13,16–20). However, detailed analysis ofthese studies shows that they have typically focused onthe study of BM CD34þ hematopoietic progenitor cells(HPC) (21–25) and/or a few major compartments ofmaturing neutrophil, monocytic and to lower extent,also erythroid and megakaryocytic cells (19–26). In addi-tion, although in few of these reports immunopheno-typic scores (IS) have been built according to thenumber of abnormalities identified and their nature(13,19,23), such scores are frequently based on rela-tively subjective criteria (13) and/or the analysis of arestricted number of BM cell populations (15,23,27).Finally, in these studies, deviation from normal is typi-cally established on the basis of the immunophenotypicfeatures of normal plus reactive BM samples frompatients suffering from different disease conditions thatcould potentially have a variable and heterogeneouseffect on the immunophenotypic patterns of BM precur-sors and maturing myeloid cells. In contrast, no compre-hensive study has been reported so far in which a

scoring system based on objective immunophenotypiccriteria is used to rank the progressively altered patternsof antigen expression observed in BM compartments ofmaturing lymphoid and myeloid cells committed intoboth major and minor hematopoietic cell lineages inMDS and in reactive BM samples, taking only normaladult BM as the reference.

In this pilot study, we provide a comprehensive analy-sis of the distribution and immunophenotypic patternsof different maturation-associated compartments ofimmature-, neutrophil-, monocytic-, erythroid-, mast cell-,plasmacytoid dendritic cell (pDC)-, basophil-, and B-lymphoid-committed hematopoietic BM cells in a rela-tively limited series of 56 consecutive patients with MDSinvestigated at diagnosis, compared with a group of 20normal adult BM samples. In addition, a third group of20 reactive BM samples corresponding to cases present-ing with cytopenias associated with non-clonal hemato-poiesis in which differential diagnosis with MDS wasrequired was also studied in parallel. On the basis of thenumber and severity of the alterations detected withrespect to normal BM, an objective IS was built thatallows clear-cut discrimination between MDS and bothnormal and reactive BM samples. Of note, the IS pro-gressively increased from low- to high-grade MDS andemerged as an independent prognostic factor for overallsurvival.

MATERIALS AND METHODS

Patients, Controls and Samples

A total of 56 untreated patients (35 men and 21women) with a mean age of 69 years (range, 31–85years), newly diagnosed with MDS, were studied.According to the World Health Organization (WHO) cri-teria (3,28), patients were classified as follows: RA, 9cases; RCMD, 10; RAEB-1, 10; RAEB-2, 13; myelodysplas-tic/myeloproliferative disorder (MD/MPD), 11 and; 5q�

syndrome, 3 cases. Following the International Prognos-tic Scoring System (IPSS) (6), 13 cases were classified aslow risk (LOW-R) MDS, 24 as intermediate-1 (INT-1-R),11 as intermediate-2 (INT-2-R), and four cases as highrisk (HIGH-R) MDS; in the remaining four patients, ei-ther metaphases could not be obtained from cultures orcytogenetic data was not available. In every patient, anEDTA-anticoagulated BM sample was obtained at diagno-sis for further multiparameter flow cytometry immuno-phenotypic studies. At the moment of closing this study,

PHENOTYPE IN MDS AND SURVIVAL 155

Cytometry Part B: Clinical Cytometry

17 patients had died and 39 remained alive with a me-dian overall survival of 15 months for the whole series.

In parallel, 40 freshly obtained, EDTA-anticoagulatednormal (n ¼ 20) and reactive (n ¼ 20) BM samplesfrom an identical number of individuals (median age, 64years; range, 46–79 years) were collected at the Univer-sity Hospital of Salamanca (Spain). Normal BM sampleswere obtained from healthy donors and individualsundergoing orthopedic surgery, whereas reactive sam-ples corresponded to patients with carential and megalo-blastic anemias and other toxic (e.g., drug-inducedcytopenias) or reactive cytopenias (e.g., idiopathicthrombopenia purpura) and infection-associated leuko-penias. None of the reactive samples showed clonalhematopoiesis, based on the absence of MDS-associatedcytogenetic abnormalities—e.g., trisomy 8, �7/7q�, �5/5q�, del(20q) or—Y- as assessed by fluorescence in situhybridization (FISH) and/or a non-clonal pattern of inac-tivation of chromosome X- negative human androgen re-ceptor assay (HUMARA) test—in FACS-purified maturingneutrophils, monocytic cells, nucleated red blood cells(NRBC), CD34þ HPC, and mature lymphocytes. All BMsamples were obtained after informed consent was givenby each individual according to the recommendations ofthe local Ethics Committee and studied within the first18 hours after they were obtained. In case they wereshipped from another center, they were packed at roomtemperature isolated from external temperature.

Immunophenotypic Studies and Flow Cytometric Score

Whole BM samples (2 � 106 cells in 100 lL/test)were stained for cell surface markers using a stain-lyse-and-then-wash direct immunofluorescence technique,previously described in detail (29,30). In addition, intra-cellular—nuclear (n) and cytoplasmic (Cy)—stainingswere performed after cell fixation and permeabilization,using the Fix and Perm reagent kit (Invitrogen, Carlsbad,CA). The following combinations of monoclonal antibod-ies (MAb) in four color stainings—fluorescein isothiocya-nate (FITC)/phycoerythrin (PE)/peridinin chlorophyllprotein (PerCPCy5.5)/allophycocyanin (APC)—were sys-tematically used: HLA-DR/CD117/CD45/CD34; HLA-DR/CD123/CD45/CD34; CD11b/CD13/CD45/CD34; CD61/CD33/CD45/CD34; nTdT/CyMPO/CD45/CD34; CD15/CD16/CD45/CD34; CD19/CyCD79a/CD45/CD34; CD65/7.1/CD45/CD34; CD36/CD64/CD45/CD34-CD14, IREM-2/CD14/CD45/CD34, CD2/CD56/CD45/CD34, CD71/CD235a/CD45/CD34, and CyCD3/CD7/CD45/CD34. Thespecificity and source of each reagent have been previ-ously described in detail (25).

Immediately after staining, sample aliquots were meas-ured in a FACSCalibur flow cytometer (Becton DickinsonBiosciences (BDB), San Jose, CA) using the CellQUESTsoftware program (BDB) for a total of 3 � 104 eventscorresponding to the whole BM cellularity, per samplealiquot. For data analysis, the INFINICYTTM softwareprogram (Cytognos SL, Salamanca, Spain) was used. Inbrief, total CD34þ BM precursors were identified andcounted according to their light scatter characteristics—

forward light scatter (FSC) and sideward light scatter(SSC)—their positivity for CD34 and dim CD45 expres-sion following the ISHAGE guidelines (31), as describedin detail elsewhere (25) (Figs. 1A and 1B). In addition,CD117þ/CD34� precursors were identified in a SSC ver-sus CD117 bivariate dot plot histogram after excludingCD34þ HPC, as exemplified in Figure 1C. AmongCD117þ/CD34� precursors, the following subsets wereidentified (Fig. 1D): (a) neutrophil-committed precursors(CD45lo/SSClo/int); (b) erythroid-committed precursors(CD45�/SSCvery-lo); and (c) maturing CD34� mast cellswere identified as those CD45þ events being CD117hi

with heterogeneous HLA-DR expression (HLA-DR� to þ;Fig. 1E); finally, in MDS cases, accumulation of abnormalCD117þ/CD34�/CD45lo/SSClo cells was also frequentlydetected. In addition to CD34þ and/or CD117þ cells, thefollowing subsets of CD34�/CD117� hematopoietic BMcells were identified: (a) neutrophil- (CD45lo/SSCint/hi), (b)erythroid- (CD45�/SSCvery-lo), (c) monocytic- (CD36�/hi/CD64hi/CD45int/hi/CD14low/hi), (d) B-cell- (CD45lo/SSCvery-lo/CD19þ/CyCD79aþ), (e) plasmacytoid dendritic cell- (pDC)(HLA-DRhi/CD123hi/CD45int), and (f) basophil-maturingcells (HLA-DR�/CD123hi/CD45int) (Fig. 1). The specificimmunophenotypic characteristics of mature lympho-cytes were taken as a standard to define the relativeposition of the different compartments of BM precur-sors in a CD45 versus SSC dot plot and to establish cut-off levels for defining the lower expression of myeloid-associated antigens.

Among CD34� neutrophil lineage cells, four differentstages of increasing maturation were defined on the ba-sis of the reactivity for the CD11b and CD13 antigens(Fig. 1G): stage I, CD13hi/CD11b� myeloblasts; stage II,CD13lo/int/CD11b� promyelocytes; stage III, CD13lo/int/CD11bþ myelocytes and metamyelocytes and; stage IV,CD13hi/CD11bþ bands and mature neutrophils. In turn,mature monocytes were identified as those cells display-ing a CD36hi, CD64hi, CD45int/hi, CD14hi immunopheno-type, whereas their CD34� precursors were defined asbeing CD36�/lo/CD64hi/CD45int/CD14lo/int (Fig. 1H).

MDS and reactive BM samples were prospectively ana-lyzed blinded to any clinical or diagnostic data. For eachantigen, results were expressed as percentage of positivecells for a given marker and/or their mean fluorescenceintensity (MFI; arbitrary relative linear units, scaled from0 to 104) within each cell subset identified. The pres-ence of aberrant phenotypes and/or other maturation-associated abnormalities were defined for each BM cellpopulation based on numerical and phenotypic devia-tions greater than the mean � 2 standard deviations(SD) from the normal BM profiles. Accordingly, for eachindividual immunophenotypic variable analyzed (n ¼83) in each BM sample, a score of 0, 0.5, 1, or 2 wasgiven when the value obtained was within the mean �2 SD, between the mean � 2 SD and the mean � 3 SD,between the mean � 3 SD and the mean � 4 SD, andover the mean � 4 SD of the values found for that pa-rameter among the normal BM samples analyzed, respec-tively. On the basis of the overall score reached,

156 MATARRAZ ET AL.

Cytometry Part B: Clinical Cytometry

FIG. 1. Bivariate dot plot histograms illustrating how the different compartments of precursors, maturing and mature cells from different hemato-poietic lineages were differentially identified in a normal bone marrow (BM) sample (A–H). (A) CD45 versus SSC primary gating for the identificationof most cell populations corresponding to different hematopoietic cell lineages (ungated events). (B) Specific secondary gating for CD34þ cells isillustrated (black dots). Black and dark blue dots in (C) and (D) represent CD34þ hematopoietic precursors and CD117þ/CD34� BM cells, respec-tively, including the compartments of neutrophil- and erythroid-committed precursors contained among CD117þ/CD34� cells (D). Cells maturing tothe mast cell lineage are identified as red dots in (E). (F) Secondary gates used for the identification of maturing basophils (red dots) and plasmacy-toid dendritic cells (pDC; green dots). (G) Gating strategy used for the identification of the different subsets of CD34� maturing neutrophils—CD13hi/CD11b�myeloblasts (stage I); CD13lo/CD11b� promyelocytes (stage II); CD13lo/CD11bþ myelocytes and metamyelocytes (stage III); andCD13hi/CD11bþ bands/mature neutrophils (stage IV). (H) Secondary gates used to identify different maturation compartments of monocytic (browndots) and erythroid (red dots) lineage cells. (A and G) A scale of colors from blue/green to brown represents increasing cell density within each popu-lation of BM cells. HPC, hematopoietic progenitor and precursor cells; NRBC, nucleated red blood cells.

PHENOTYPE IN MDS AND SURVIVAL 157

Cytometry Part B: Clinical Cytometry

individual patients with MDS were phenotypically classi-fied as mild (score <10), intermediate (score �10 and<20), and severely altered (score �20).

Immunophenotypic Variables under Study

Up to 83 different immunophenotypic variables wereinvestigated in each case: (a) immature cell compart-ments: % of CD34þ, % of CD34�/CD117þ, % of CD34�/CD117þ neutrophil- and % of CD34�/CD117þ erythroid-committed precursors, % of CD34� B-cells and expres-sion (MFI) of CD19 and Cy-CD79a on the latter popula-tion; (b) maturing neutrophils: % of CD34� neutrophillineage cells and MFI of FSC, SSC, CD45, CD15, CD16,CyMPO, CD11b, CD13, CD33, CD64, CD65, % ofCD16hi, % of CD64hi and % of CD14þ and % of CD56þ

aberrant cells together with the % of neutrophil cells inthe maturation stages I, II, III and IV (see above) andMFI of CD45, SSC, CD11b and CD13 within each ofthese four neutrophil maturation stages; (c) monocyticmaturation: % of monocytic cells, % of monocytic pre-cursors (CD14�/int), % of mature (CD14hi) monocytesand MFI of FSC, SSC, CD14, CD36, CD45 and CD64within each of these subsets together with overall reac-tivity of monocytic cells for CD11b, CD13, CD15, CD33,CD65, CyMPO, HLA-DR, % of IREM-2þ cells togetherwith the % of CD2 and % of CD56 aberrant monocyticcells; (d) erythroid maturation: % of NRBC, % of aberrantCD36�/lo cells and MFI of CD71, CD235a and CD36 onNRBC compartment; (e) basophil and pDC: % of baso-phils, % of pDC and expression levels (MFI) of CD123on both groups of cells.

Conventional Karyotyping, FISH, and HUMARA Studies

Cytogenetic analysis of BM samples was performedaccording to standard procedures (32) and interpretedusing the International System for Cytogenetic Nomen-clature criteria (33). In addition, interphase FISH (iFISH)studies were systematically performed on each MDS BMsample, as previously reported (34). In these cases, thefollowing chromosome probes purchased from Vysis Inc(Downers Grove, IL) were systematically used in doublestainings for the detection of the most frequent recur-rent abnormalities: (1) LSI D5S23, D5S71 SpectrumGreen (SG)/LSI EGFR Spectrum Orange (SO) probe com-bination for chromosome 5; (2) LSI D7S486 (7q31) SO/CEP 7 SG probes for chromosome 7; (3) CEP 8 (D8Z2)SO/CEP Y (DYZ1) SG probes for chromosomes 8 and Y,respectively; (4) LSI D20S108 (20q12) SO probe forchromosome 20. Investigation of the pattern of inactiva-tion of chromosome X was analyzed in FACS-purifiedcells (please see above) as described elsewhere (35).

Statistical Methods

For all variables under study, their mean values andSD, median and range were calculated using the SPSSsoftware (SPSS 10.0, Chicago, IL). Comparisons betweentwo or more groups were made using the v2, for cate-gorical variables; for continuous variables, the Student t

(for parametric data) and either the Mann–Whitney U or

the Kruskal–Wallis tests (nonparametric data) wereused. Survival curves were plotted according to themethod of Kaplan and Meier (36), and the statistical sig-nificance of the differences observed in survival was cal-culated with the log-rank test. On the basis of thosevariables showing a significant impact on overall survivalin the univariate analysis, a multivariate Cox propor-tional-hazards model was constructed with those varia-bles showing independent predictive value; inclusion inthe final model was determined by a backward stepwiseprocess. P values <0.05 were considered to be associ-ated with statistical significance.

RESULTS

Immunophenotypic Features of BM Cell Populations inReactive BM Samples

The analysis of reactive BM samples (n ¼ 20) revealedthe presence of increased numbers over the mean � 2SD of both CD34þ and CD34�/CD117þ cells (P ¼ 0.01)(Table 1) together with increased percentages ofCD13hi/CD11b� myeloblasts, stage I maturing neutrophilprecursors (P < 0.001; Table 1). Of note, a trend towardlower SSC characteristics and decreased expression ofCD13, CD15, CD11b in neutrophils and monocytic cellsand of CD71 and CD235a in NRBC precursors wasobserved among reactive BM samples but except forCD71 and CD11b (P � 0.05), differences did not reachstatistical significance. In turn, aberrant expression oflymphoid-associated markers was never detected (Tables1 and 2).

Distribution of Maturing BM Cell Compartments inPatients with MDS

Overall, increased percentages of BM CD34þ cells andof CD34�CD117þ maturing precursors were found inaround half of all patients with MDS (51% of the caseseach; P � 0.001); such increase in precursor cells wasassociated with the presence of phenotypically aberrantCD34�/CD117þ cells in 33% of cases (P ¼ 0.003) (Table1; Fig. 2). In addition, CD34�CD117þ neutrophil- anderythroid-committed precursors were also altered in 45%and 54% of all patients with MDS (P � 0.001), a highproportion of cases showing decreased numbers of bothcell subsets (27% and 47%, respectively; P � 0.001)(Table 1). Of note, this was associated with an overalldecreased percentage of maturing CD34� neutrophils in33% of the patients (P ¼ 0.006), but either normal orincreased (22% of cases) CD34�/CD117� maturingNRBC (P ¼ 0.08) (Table 1) particularly among RCMDpatients (P ¼ 0.01; data not shown). Interestingly,within the neutrophil compartment a maturation block-ade, reflected by increasing numbers of the more imma-ture CD34� neutrophil lineage cells (stages I and II; P �0.01) and decreased percentages of the two moremature subsets (stages III and IV; P � 0.02) wasdetected (Table 1). Moreover, the overall number of BMB-cells was also decreased among patients with MDS (P¼ 0.02), particularly among the more advanced stages of

158 MATARRAZ ET AL.

Cytometry Part B: Clinical Cytometry

Tab

le1

Distribution

ofTotal

CD34þBM

Cells

andof

DifferentCom

partm

ents

ofBM

CD34�Hem

atop

oietic

Precu

rsorsan

dMature

Cells

(leftpan

el)in

Patients

WithMDSversus

Normal/ReactiveBon

eMarrow(BM)IncludingtheFrequen

cyof

Cases

DisplayingAbnormally

Increased/Decreased

Percentagesof

BM

Precu

rsors(rightpan

el)Within

Specific

Subsets

ofCD34�HPC

BM

cell

subse

ts

Dis

trib

uti

onof

cell

pop

ula

tion

s(%

)P

erce

nt

ofal

tere

dca

ses

(incr

ease

d/d

ecre

ased

)a

Nor

mal

BM

(N¼

20

)R

eact

ive

BM

(N¼

20

)M

DS

(N¼

56

)P

bR

eact

ive

BM

(N¼

20

)P

cM

DS

(N¼

56

)P

b

Tot

alC

D3

4þ

cells

0.8

�0

.3%

(0.2

–1.6

%)

1�

0.3

%(0

.6–1

.6%

)d2

.5�

3%

(0.2

–15

%)

<0

.00

12

5%

/—0

.02

51

/—(5

1%

)<

0.0

01

Mat

uri

ng

CD

34�C

D1

17þ

cells

—/—

(0%

)

Tot

alC

D3

4�/C

D1

17þ

pre

curs

ors

1�

0.8

%(0

–2%

)2�

1%

(0–4

%)d

4�

4%

(0–2

0%

)<

0.0

01

25

%/—

0.0

25

1/—

(51

%)

0.0

01

Per

cent

ofer

ythro

idpre

curs

orse

36�

10

%(2

2–5

4%

)3

4�

11

%(2

2–6

1%

)2

3�

20

%(0

–70

%)

<0

.00

1—

/—(0

%)

NS

7/4

7%

(54

%)

<0

.00

1

Per

cent

ofneu

trop

hil

pre

curs

orse

62�

11

%(4

5–7

8%

)6

5�

12

%(3

8–8

0%

)5

4�

30

%(0

–10

0%

)<

0.0

01

—/—

(0%

)N

S1

8/2

7%

(45

%)

0.0

01

Per

cent

ofab

nor

mal

CD

34�/C

D1

17þ/S

SC

lo

cellse

0�

0%

0�

0%

17�

31

%(0

–10

0%

)0

.00

3—

/—(0

%)

NS

33

/—(3

3%

)0

.00

3

Mat

ure

and

mat

uri

ng

CD

34�

cells

Neu

trop

hil

linea

ge5

9�

10

%(4

6–7

4%

)5

3�

10

%(4

0–7

3%

)d4

5�

18

%(3

–80

%)

0.0

01

—/—

(0%

)N

S2

/33

%(3

5%

)0

.00

6S

tage

I2�

1%

(0.6

–5%

)4�

2%

(1.5

–8%

)6�

7%

(0–3

8%

)0

.01

58

%/—

<0

.00

12

8%

/—(2

8%

)0

.00

4S

tage

II1

1�

5%

(3–2

5%

)1

7�

7%

(6–3

0%

)2

1�

12

%(4

–55

%)

0.0

02

—/—

(0%

)N

S4

0%

/—(4

0%

)<

0.0

01

Sta

geII

I3

2�

8%

(12

–44

%)

36�

6%

(30

–50

%)

27�

13

%(0

–65

%)

0.0

2—

/—(0

%)

NS

2/2

5%

(27

%)

0.0

2S

tage

IV5

1�

11

%(3

5–5

6%

)4

1�

9%

(18

–55

%)

43�

17

%(2

–84

%)

0.0

2—

/—(0

%)

NS

7/2

1%

(28

%)

0.0

1C

D1

17�

Mon

ocyt

iclinea

gece

lls

4�

1%

(2–6

%)

3.5

�1

%(3

–5%

)6

.3�

6.5

(0-3

0)

<0

.00

1—

/—(0

%)

NS

38

/18

%(5

6%

)<

0.0

01

Mon

ocyt

icpre

curs

ors

1.6

�0

.8%

(0.6

–4%

)1�

0.5

%(0

.3–2

%)

3.5

�3

%(0

–13

%)

<0

.00

18

%/—

NS

36

/11

%(4

7%

)<

0.0

01

Mat

ure

mon

ocyt

es2

.5�

1%

(1–4

.5%

)3�

0.7

%(1

.5–4

%)

3�

4%

(0–1

9%

)0

.00

2—

/—(0

%)

NS

18

/15

%(3

3%

)0

.00

8C

D1

17�

Nucl

eate

dre

dblo

odce

lls

15�

7%

(5–2

9%

)2

0�

10

%(5

–40

%)

21�

13

%(4

–70

%)

0.0

8—

/—(0

%)

NS

22

/—(2

2%

)0

.08

Tot

alB

cells

3�

2%

(0.0

3–1

0%

)2

.7�

1.5

(0.0

5–9

%)

1.5

�2

%(0

–8%

)0

.02

—/—

(0%

)N

S7

/2%

0.7

CD

34�CD117�B-cell

precu

rsors

1�

0.7

%(0

.05

–2.6

%)

1.4

�1

(0.0

6–3

.5%

)0

.4�

0.6

%(0

–2%

)<

0.0

01

—/—

(0%

)N

S—

/49

%(4

9%

)<

0.0

01

Mature

CD45hi

B-lym

phocytes

1.5

�0

.8%

(0.3

–3%

)1

.6�

1(0

.5–3

.5%

)1�

1.2

%(0

–6%

)0

.1—

/—(0

%)

NS

9%

/—(9

%)

0.3

Bas

ophils

0.4

�0

.7%

(0.0

5–3

%)

0.2

�0

.1%

(0.1

–0.5

%)

0.4

�0

.6%

(0–3

.5%

)0

.8—

/—(0

%)

NS

7%

/—(7

%)

0.8

Mas

tce

lls

0.0

05�

0.0

08

%(0

–0.0

2%

)0

.03�

0.0

5%

(0–0

.15

%)

0.0

06�

0.0

2%

(0–0

.1%

)0

.6—

/—(0

%)

NS

5%

/—(5

%)

0.6

Pla

smac

ytoi

dden

dri

tic

cells

0.2

�0

.1%

(0–0

.6%

)0

.3�

0.1

%(0

.2–0

.5%

)0

.2%

�0

.3%

(0–1

.4%

)0

.2—

/—(0

%)

NS

15

%/—

(15

%)

0.2

Res

ult

sex

pre

ssed

asm

ean�

1S

D(r

ange

)per

centa

geof

cells

from

the

whol

esa

mple

cellula

rity

and:

afr

equen

cyof

alte

red

case

s–m

ean

valu

esov

erth

enor

mal

mea

nplu

s(i

ncr

ease

d)

orm

inus

(dec

reas

ed)

two

stan

dar

ddev

iati

ons-

;non

eof

the

20

nor

mal

BM

sam

ple

ssh

owed

phen

otyp

icva

lues

over

the

mea

n�

2S

D.

bM

DS

vsnor

mal

BM

.cre

acti

vevs

nor

mal

BM

.dp<

0.0

5vs

nor

mal

BM

.eto

tal

CD

34�/C

D1

17þ

cells.

pD

C,

pla

smac

ytoi

dden

dri

tic

cells;

SS

C,

sidew

ard

ligh

tsc

atte

r;N

S,

not

stat

isti

cally

sign

ifica

nt;

MD

S,

mye

lodys

pla

stic

syndro

mes

;N

/R,

nor

mal

and

reac

tive

;B

M,

bon

em

arro

w;

HP

C,

hem

atop

oiet

icpre

curs

orce

lls.

AQ4

Cytometry Part B: Clinical Cytometry

PHENOTYPE IN MDS AND SURVIVAL 159

Tab

le2

Phen

otypic

Characteristicsof

DifferentCom

partm

ents

ofCD34�Hem

atop

oietic

Precu

rsorsan

dMature

Cells

(leftpan

el)in

Patients

WithMDSversusNormal

andReactive

BM

IncludingtheFrequen

cyof

Cases

DisplayingAbnormally

Increased/Decreased

Expressionof

Individual

Antigens(rightpan

el)

Cel

lpop

ula

tion

san

dal

tere

dphen

otyp

icm

arke

rs

Imm

unop

hen

otyp

icch

arac

teri

stic

sP

erce

nt

ofal

tere

dca

ses

(incr

ease

d/d

ecre

ased

)a

Nor

mal

BM

(N¼

20

)R

eact

ive

BM

(N¼

20

)M

DS

(N¼

56

)P

bN

orm

alB

M(N

¼2

0)

Rea

ctiv

eB

M(N

¼2

0)

MD

S(N

¼5

6)

Pb

Mat

uri

ng

neu

trop

hils

FS

CM

I4

62�

80

(35

0–6

00

)4

50�

45

(40

0–5

00

)4

25�

11

0(1

90

–80

0)

0.0

2—

/—(0

%)

—/—

(0%

)—

/20

%(2

0%

)0

.03

SS

CM

I5

32�

10

0(2

70

–70

0)

29

5�

11

2(2

00

–60

0)

29

0�

14

0(1

10

–75

0)

<0

.00

1—

/-(0

%)

—/8

%—

/43

%(4

3%

)<

0.0

01

CD

45

MFI

16

5�

60

(70

–27

0)

14

4�

80

(50

–33

0)

20

0�

13

0(3

0–6

50

)0

.01

—/—

(0%

)—

/—(0

%)

21

%/2

%(2

3%

)0

.04

CD

11

bM

FI

45

0�

16

0(1

00

–72

0)

30

0�

15

0(9

0–5

50

)c3

30�

20

0(4

6–8

80

)0

.02

—/—

(0%

)—

/—(0

%)

4%

/2%

(6%

)0

.5C

D1

3M

FI

50

0�

23

0(2

50

–11

00

)4

00�

19

0(6

3–6

25

)3

70�

31

0(1

5–2

00

0)

0.0

05

—/—

(0%

)8

%/—

13

%/2

%(1

5%

)0

.7C

D3

3M

FI

16

0�

11

0(5

0–6

00

)1

10�

50

(30

–18

0)

13

5�

98

(25

–48

0)

0.0

2—

/—(0

%)

—/—

(0%

)7

%/1

3%

(20

%)

0.0

7C

D1

5M

FI

48

00�

20

00

(70

0–8

20

0)

38

00�

25

00

(95

0–8

40

0)

30

00�

20

00

(25

0–8

90

0)

0.0

06

—/—

(0%

)—

/—(0

%)

—/4

1%

(41

%)

0.0

06

CD

64

MFI

20

0�

60

(80

–30

0)

20

0�

90

(80

–40

0)

22

0�

20

0(1

0–1

00

0)

0.0

1—

/—(0

%)

16

%/—

18

%/1

4%

(32

%)

0.0

3C

D6

5M

FI

80

0�

40

0(1

80

–20

00

)7

50�

74

0(8

0–2

10

0)

46

5�

40

0(2

0–2

00

0)

0.0

05

—/—

(0%

)—

/16

%4

%/4

3%

(47

%)

0.0

6C

yMP

OM

FI

74

0�

44

0(2

00

–18

00

)6

30�

62

0(7

0–2

00

0)

72

0�

10

00

(26

–58

00

)0

.06

—/—

(0%

)—

/—(0

%)

9%

/39

%(4

8%

)0

.09

Per

cent

ofC

D1

6hice

lls

60�

10

%(5

0–8

8%

)5

0�

16

%(2

0–7

0%

)4

4�

22

%(1

–94

%)

0.0

08

—/—

(0%

)8

%/—

4%

/39

%(4

3%

)0

.00

8P

erce

nt

ofC

D6

4hice

lls

50�

10

%(2

0–6

5%

)5

5�

10

%(3

6–7

7%

)4

5�

30

%(0

–10

0%

)0

.00

7—

/—(0

%)

8%

/—1

4%

/27

%(4

1%

)0

.01

Per

cent

ofab

erra

nt

CD

56þ

cells

0�

0%

0�

0%

6�

10

%(0

–44

%)

0.0

01

—/—

(0%

)—

/—(0

%)

43

%/—

(43

%)

0.0

01

Per

cent

ofab

erra

nt

CD

14þ

cells

0�

0%

0�

0%

15�

23

%(0

–10

0%

)0

.00

1—

/—(0

%)

—/—

(0%

)4

1%

/—(4

1%

)0

.00

1

Mon

ocyt

iclinea

ge—

/—(0

%)

FS

CM

I3

80�

50

(29

0–4

60

)3

40�

80

(28

0–4

60

)3

60�

90

(22

0–6

00

)<

0.0

01

—/—

(0%

)—

/—(0

%)

—/2

0%

(20

%)

0.0

1S

SC

MI

30

0�

98

(10

0–5

00

)1

51�

82

(10

0–3

80

)1

50�

70

(60

–44

0)

<0

.00

1—

/—(0

%)

—/—

(0%

)—

/30

%(3

0%

)<

0.0

01

CD

11

bM

FI

45

0�

20

0(6

0–8

50

)3

00�

19

0(6

4–6

50

)c3

70�

27

0(3

5–1

30

0)

0.0

6—

/—(0

%)

—/—

(0%

)5

%/2

5%

(30

%)

0.5

CD

13

MFI

10

00�

60

0(1

90

–26

00

)8

00�

50

0(8

0–1

60

0)

78

0�

67

0(4

2–3

30

0)

0.0

3—

/—(0

%)

—/—

(0%

)5

%/3

4%

(39

%)

0.0

6C

D1

4M

FI

85

0�

45

0(3

00

–22

00

)7

50�

25

0(3

70

–11

0)

70

0�

54

0(1

00

–33

00

)0

.02

—/—

(0%

)—

/—(0

%)

—/2

0%

(20

%)

0.0

2C

D3

6M

FI

85

0�

17

0(5

00

–11

50

)6

40�

30

0(3

40

–13

00

)7

00�

30

0(9

0–1

30

0)

0.0

02

—/—

(0%

)—

/—(0

%)

10

%/2

1%

(31

%)

0.0

08

CD

64

MFI

14

00�

50

0(5

40

–25

00

)1

34

0�

40

0(5

00

–18

00

)1

20

0�

80

0(7

4–4

00

0)

0.0

09

—/—

(0%

)—

/—(0

%)

7%

/16

%(2

3%

)0

.03

Per

cent

ofIR

EM

-2þ

cells

60�

10

%(4

0–8

0%

)5

4�

8%

(50

–70

%)

50�

23

%(0

–10

0%

)0

.02

—/—

(0%

)—

/—(0

%)

4%

/18

%(2

2%

)0

.05

Per

cent

ofab

erra

nt

CD

56þ

cells

0�

0%

0�

0%

30�

34

%(0

–80

0%

)<

0.0

01

—/—

(0%

)—

/—(0

%)

48

%/—

(48

%)

<0

.00

1

Per

cent

ofab

erra

nt

CD

2þ

cells

0�

0%

0�

0%

35�

50

%(0

–10

0%

)0

.00

2—

/—(0

%)

—/—

(0%

)3

2%

/—(3

2%

)0

.00

2

Ery

thro

idlinea

ge—

/—(0

%)

CD

36

MFI

76

0�

16

0(5

40

–11

00

)6

70�

90

(50

0–8

00

)6

00�

33

0(1

50

–15

00

)<

0.0

01

—/—

(0%

)—

/—(0

%)

10

%/3

0%

(40

%)

0.0

01

Per

cent

ofab

erra

nt

CD

36

—/lo

cells

0�

0%

0�

0%

19�

20

%(0

–10

0%

)<

0.0

01

—/—

(0%

)—

/—(0

%)

71

%/—

(71

%)

<0

.00

1

CD

71

MFI

80

0�

34

0(2

60

–14

00

)4

25�

75

(34

0–5

60

)c5

20�

36

0(2

0–1

40

0)

0.0

05

—/—

(0%

)—

/—(0

%)

5%

/2%

(7%

)0

.8C

D2

35

aM

FI

11

00�

67

0(1

40

–26

00

)6

70�

18

0(5

00

–11

00

)8

00�

62

0(2

5–2

50

0)

0.0

5—

/—(0

%)

—/—

(0%

)7

%/—

(7%

)0

.6B

-cel

llinea

geC

y-C

D7

9a

MFI

onC

D3

4�

pre

curs

ors

21

0�

90

(10

0–4

40

)2

10�

16

0(9

–52

5)

18

0�

14

0(5

–62

0)

0.0

3—

/—(0

%)

—/—

(0%

)—

/19

(19

%)

0.0

3

Res

ult

sex

pre

ssed

asm

ean�

one

stan

dar

ddev

iati

on(a

nd

range

bet

wee

nbra

cket

s)or

as:

aper

centa

geof

alte

red

case

sw

ith

incr

ease

d/d

ecre

ased

(tot

alal

tere

d)

valu

es.

No

case

was

det

ecte

dsh

owin

gab

nor

mal

MFI

valu

esfo

rC

D1

6on

neu

trop

hils,

for

CD

45

,C

D1

5,

Cy-

MP

O,

HLA

-DR

,C

D6

5an

dC

D3

3on

mon

ocyt

icce

lls

and

for

CD

19

,cy

CD

3,

CD

12

3,

CD

61

and

7.1

mar

kers

for

any

ofth

eB

Mce

llco

mpar

tmen

tsan

alyz

ed.

MFI,

mea

nfluor

es-

cence

inte

nsi

ty(a

rbit

rary

fluor

esce

nce

unit

ssc

aled

from

0to

10

4);

MI,

mea

nin

tensi

ty(a

rbit

rary

unit

ssc

aled

from

0to

10

23

);B

M,

bon

em

arro

w;

MD

S,

mye

lodys

pla

stic

syndro

me.

bM

DS

vsnor

mal

BM

.cP<

0.0

5vs

nor

mal

BM

.

160 MATARRAZ ET AL.

Cytometry Part B: Clinical Cytometry

the disease—INT-1, INT-2 and patients with HIGH MDS(P ¼ 0.01; data not shown). This was associated withdecreased numbers of CD34� B-cell precursors detectedin around half of all patients with MDS (49%; P < 0.001)(Table 1). Notably, maturing CD34�/CD117� monocyticBM cells were frequently altered (56%), 38% of all MDScases showing increased numbers of these cells associ-ated with an abnormally high monocytic precursor/mature monocyte ratio, particularly among RAEB-2 andINT-2-R patients (P � 0.001) (Table 1; Fig. 3).

In contrast, no significant differences were detectedas regards the distribution of mature B-lymphocytes,basophil-, mast cell-, and pDC-lineage cells in MDS ver-sus normal BM (Table 1).

Immunophenotypic Features of BM Cell Populations inPatients with MDS

In addition to the numerical abnormalities describedabove, multiple phenotypic changes were detected inthe different compartments of maturing BM cells inpatients with MDS (Table 2). Accordingly, maturing neu-trophils showed decreased light scatter characteristics inaround half of all MDS patients (SSC: 43%, P < 0.001and; FSC: 20%, P ¼ 0.03). Of note, the most relevantphenotypic alterations detected in this cell compart-ment—maturing neutrophils—included abnormally lowreactivity for CD15, CD65, and CyMPO observed inbetween 39% and 43% of all patients with MDS (P �0.09), together with decreased numbers of CD16hi

maturing neutrophils (39% of all patients with MDS; P ¼0.008) (Table 2). In turn, increased numbers of maturing

neutrophils aberrantly expressing CD56 (43%) and/orCD14 (41%) were also frequently detected in patientswith MDS (P ¼ 0.001), whereas increased levels of

FIG. 2. Dot plot histograms illustrating the distribution of normal CD117þ and/or CD34þ cells in a normal bone marrow (A) versus aberrantCD117þ/CD34� precursors in a BM sample from a patient with MDS (B) and in overlayed data files using the merge function of the Infinicyt software(C–E). Neutrophil and erythroid CD117þ/CD34� precursors are colored blue; CD34þ cells are black and abnormal CD34�/CD117þ/CD45lo/SSClo arecolored red.

FIG. 3. Ratio between the percentage of CD64hi, CD36�/lo monocyticprecursors and CD64hi, CD36þ mature monocytes in MDS versus bothnormal (N) and reactive (R) bone marrow (BM) samples groupedaccording to the World Health Organization (WHO) classification (A)and the International Prognostic Scoring System (IPSS) (B). Notched-boxes represent 25th and 75th percentile values; the line in the mid-dle and vertical lines correspond to the median value and 95% confi-dence intervals, respectively. RA, refractory anemia; RCMD, refractorycytopenia with multilineage dysplasia; RAEB, refractory anemia withexcess of blasts; MD/MPD, myelodysplastic/myeloproliferative disorder;5q�, 5q� syndrome; LOW-R, low risk MDS; INT-R, intermediate riskMDS; HIGH-R, high risk MDS. * and �, P < 0.05; ** and ��, P <0.03 for comparisons between MDS and both N and R BM samples,respectively.

PHENOTYPE IN MDS AND SURVIVAL 161

Cytometry Part B: Clinical Cytometry

Tab

le3

Enumerationof

Immunop

hen

otypic

Alterationsan

dFlowCytom

etry

Immunop

hen

otypic

Scoresin

Normal/Rea

ctive(N

/R)Bon

eMarrow(BM)versusMDSPatients

Grouped

Accordingto

theWorld

HealthOrgan

ization(W

HO)Classification

andtheInternational

Progn

osticScoringSystem

(IPSS)

BM

cell

com

par

tmen

t(N

o.phen

otyp

icpar

amet

ers

anal

yzed

per

BM

sam

ple

)

WH

Osu

bty

pe

IPS

Sca

tego

ry

Inte

rmed

iate

risk

Nor

mal

BM

(N¼

20

)

Rea

ctiv

eB

M(N

¼2

0)

5q�

(N¼

3)

RA

(N¼

9)

RC

MD

(N¼

10

)R

AE

B-1

(N¼

10

)R

AE

B-2

(N¼

13

)M

D/M

PD

(N¼

11

)Tot

alM

DS

(N¼

56

)Low

risk

(N¼

14

)IN

T-1

(N¼

23

)IN

T-2

(N¼

11

)H

IGH

risk

(N¼

4)

Imm

ature

cell

com

par

tmen

ts(n

¼7

)

0�

00

.09�

0.3

(0–1

)1

.6�

1.5

(0–3

)1

.8�

1.2

(0–4

)1

.5�

1(0

–4)

3.6

�1

.5(1

–6)

5�

1(4

–7)

3�

1(1

–4)

3�

2(0

–7)

1.7

�1

.4(0

–4)

2.5

�1

.5(0

–5)

5�

1(3

–7)

4.5

�1

(3–5

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3).

162 MATARRAZ ET AL.

Cytometry Part B: Clinical Cytometry

CD45 were found in 21% of the cases (P ¼ 0.04) (Table2). Overall reactivity for CD11b and CD13 was alsofound to be decreased among patients with MDS (P �0.02) (Table 2).

Regarding BM monocytic lineage cells, the most fre-quently altered phenotypes consisted of decreased SSC(P < 0.001) and decreased reactivity for CD13, CD14,CD36, and CD64 detected in between 16% and 34% ofall MDS cases (P � 0.06); in addition, abnormally lowpercentages of mature monocytes (as reflected by alower proportion of IREM-2þ cells) were identified in18% of the cases (P ¼ 0.05) (Table 2). In addition,monocytic lineage cells from patients with MDS also dis-played aberrant expression of the CD56 and/or CD2lymphoid-associated antigens in 48% and 32% of thecases, respectively (P � 0.01; Table 2). NRBC also exhib-ited altered phenotypic profiles, which mainly consistedof decreased reactivity for CD36 found in up to 71% ofall MDS patients (P < 0.001) and for CD71 and CD235a(P � 0.05) (Table 2). Of note, abnormally low levels ofCyCD79a among CD34� B-cell precursors were found in19% of all patients with MDS (P ¼ 0.03; Table 2).

IS and its Relationship with the IPSS and WHO Subtypesof MDS and other Disease Features

According to the overall IS of individual BM samples,a clear discrimination between MDS (score >1.5) andboth normal and reactive BM (score of �1.5) wasobserved (Table 3). As expected, none of the 20 normalcontrol BM samples showed phenotypic abnormalitiesin the maturing myeloid, monocytic, B-cell or immatureCD34þ and CD34�/CD117þ cell compartments ana-lyzed; in turn, reactive BM samples from patients whoexhibited cytopenias and were evaluated to rule out aclonal hematologic disorder, showed some flow cytomet-ric abnormalities typically with a score of �1.5 (mean,0.6 � 0.7; range, 0–1.5). In contrast, the overall mean(�1 SD) score found among MDS cases was of 16 � 8.Of note, the mean score of MDS cases increased fromlow to high grade in MDS patients according to both theWHO classification and the IPSS (Table 3). Upon group-ing of patients with MDS according to their individualoverall IS (Table 4), a clear association was observedbetween a higher flow cytometric score and other rele-vant disease features, including a higher percentage ofcases with �2 cytopenias (P ¼ 0.04), thrombocytopenia(P ¼ 0.02) and increased cytomorphologic blast cellcounts (P < 0.001).

IS and Overall Patient Survival

Once patients with MDS were distributed accordingto their overall IS, a significant (P ¼ 0.01) impact onpatients overall survival was found with median survivalrates of 86 � 10, 37 � 0, and 21 � 14 months forpatients displaying low (<10), intermediate (10–19), andhigh (�20) IS, respectively (Fig. 4A). Upon considerationof individual immunophenotypic alterations, only thenumber of CD34þ cells (P ¼ 0.007) together with the

Tab

le4

Relationship

betweentheOverallIm

munop

hen

otypic

Score

(IS)of

Patients

WithMyelodysplastic

Syndromes

(MDS)an

dthemostRelevan

tHem

atological,Bioch

emical,

andCytogen

etic

Characteristicsof

theDisease

ISN

o.cy

topen

ias

(�2

)A

nem

ia(<

10

0g/

L)

Thro

mboc

ytop

enia

(<1

00�

10

9/L

)Leu

kopen

ia(<

15�

10

9/L

)In

crea

sed

LD

H(>

40

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/L)

Cyt

omor

phol

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stce

llco

unt

Flo

wcy

tom

etri

cC

D3

4þ

cell

count

Inte

rmed

iate

/poo

rcy

toge

net

ics

<1

0(n

¼1

6)

5/1

5(3

3%

)9

/14

(64

%)

2/1

2(1

6%

)1

2/1

4(8

5%

)1

/8(1

2%

)3

/16

(19

%)

2/1

5(1

3%

)0

/12

(0%

)�

10

and<

20

(n¼

19

)9

/19

(47

%)

11

/19

(58

%)

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9(3

7%

)1

6/1

9(8

4%

)3

/17

(18

%)

2/1

7(1

2%

)5

/19

(10

%)

2/1

9(1

0%

)�

20

(n¼

21

)1

4/2

0(7

0%

)1

1/2

0(5

5%

)1

2/2

0(6

0%

)1

6/2

0(8

0%

)6

/18

(33

%)

16

/19

(84

%)

14

/21

(67

%)

3/2

1(1

4%

)Tot

alM

DS

28

/51

(56

%)

31

/53

(58

%)

21

/51

(40

%)

44

/51

(86

%)

10

/43

(23

%)

21

/52

(40

%)

21

/56

(40

%)

5/5

2(1

0%

)P

0.0

40

.80

.02

0.9

0.4

<0

.00

1<

0.0

01

0.1

Res

ult

sex

pre

ssed

asnum

ber

ofca

ses

from

all

MD

Spat

ients

studie

dan

dper

centa

gebet

wee

nbra

cket

s.LD

H,

lact

ate

deh

ydro

genas

e.

PHENOTYPE IN MDS AND SURVIVAL 163

Cytometry Part B: Clinical Cytometry

presence of >30% abnormal CD36�/lo erythroid precur-sors (P ¼ 0.006) showed a significant impact on theoverall survival of patients with MDS (Figs. 4D and 4F).In turn, when different immunophenotypic alterationswere simultaneously considered, a significant adverseimpact on patient survival was also found for the coexis-tence of abnormal CD34�/CD117þ cells with the obser-vation of �40% CD16hi maturing neutrophils (P ¼ 0.03),>30% of abnormal CD36�/lo NRBC (P ¼ 0.02) highnumbers of CD34þ cells (P ¼ 0.02) as well as for thecoexistence of �25% CD34�/CD117þ neutrophil precur-sors in the absence of erythroid precursors amongCD34�/CD117þ cells (P ¼ 0.03) and the simultaneouspresence of >30% CD36�/lo NRBC and �40% CD16hi

neutrophils (P ¼ 0.01) (Figs. 4G–4K).Multivariate analysis of prognostic factors showed that

the IS (low versus intermediate/high) together with thepresence of both thrombocytopenia and anemia were

the only parameters showing independent predictivevalue for overall survival in MDS, with hazard ratios(95% confidence interval) of 4.6 (1.3–17), 4.9 (1.3–18),and 8.0 (1–66), respectively.

DISCUSSION

Clonal dysregulation of hematopoiesis translating intoaltered morphologic patterns of BM maturation with PBcytopenias is a hallmark of patients with MDS (37). Suchabnormalities are also associated with an altered distri-bution of different BM cell compartments and their phe-notypic characteristics (13,23,25), involving both earlyCD34þ HPC and different compartments of maturingmyeloid and B-lymphoid cells (20,23,25,38). At present,it is well established that the number and the type anddegree of severity of the alterations (e.g., hematologic,morphologic, and cytogenetic) detected, provide infor-mation about the degree of impairment of gene

FIG. 4. Impact of the immunophenotypic abnormalities and other disease features on the overall survival of patients with myelodysplastic syn-dromes (MDS) (n ¼ 56). In the overall survival curves plotted, patients with MDS are grouped according to their immunophenotypic score (A), thenumber of peripheral blood platelets (B), and hemoglobin levels (C), the percentage of BM CD34þ cells (D), the BM cytomorphologic blast cell count(E), both the percentage of CD36�/lo NRBC (F), both the percentage of CD117þ/CD34� neutrophil and erythroid precursors (G), the presence ofabnormal CD117þ/CD34� precursors and the percentage of CD16hi neutrophils (H), the relative number of CD16hi neutrophils and CD36�/lo NRBC(I), the proportion of CD36�/lo NRBC and the presence of abnormal CD117þ/CD34� precursors (J) and the percentage of both CD34þ cells andabnormal CD117þ/CD34� precursors (K). (L and M) The prognostic impact of the IPSS and WHO classifications of patients with MDS are depicted,respectively. NRBC, nucleated red blood cells; BM, bone marrow.

164 MATARRAZ ET AL.

Cytometry Part B: Clinical Cytometry

expression and deviation from normal, with a clearimpact on patient outcome; in turn, the exact value ofimmunophenotyping remains to be more preciselyestablished (13,20,23,25,38,39).

To the best of our knowledge, this is the first reportin which numerical abnormalities and aberrant immuno-phenotypic features present in different compartmentsof both major and minor subsets of maturing/matureneutrophil, erythroid, monocytic, mast cell, pDC, baso-phil and B-lymphoid BM cells, are simultaneouslyassessed in MDS versus both normal and reactive (cyto-penic) BM, where each abnormality is scored accordingto its degree of deviation from normal BM patterns. Innormal BM, all parameters analyzed showed a distribu-tion close to normal; thus, the use of the mean plus twoSD as the cut-off for normal reference ranges for allimmunophenotypic variables is statistically robustenough for reproducibility. Objective calculation of suchdeviation based on individual parameters may improvethe evaluation of the degree of severity of the pheno-typic changes observed in patients with MDS, whileexpert-based interpretation of the patterns of expressionof multiple antigens—relationship between two or moreantigens—although equally informative, might be moresubjective and equivocal (40). Because of this, in thepresent study we attempt to reduce complex immuno-phenotypic profiles into objective numbers that reflectthe distribution of specific cell compartments in the BMand their phenotypic features, evaluated in terms of MFIlevels detected for individual markers. Overall, ourresults confirm and extend on previous observations,which indicate that based on the number and severity ofthe immunophenotypic abnormalities detected, MDScould be clearly discriminated from both normal and re-active BM; as expected (16,38,41), a highly heterogene-ous spectrum of immunophenotypic abnormalities andmaturational blockades within different compartmentsof maturing myeloid and B-lymphoid cells was observedamong patients with MDS. Of note, several of the phe-notypic alterations identified in MDS (e.g., increasednumbers of CD34þ HPC and CD11b�/CD13hi neutrophilprecursors), were also detected in BM cells frompatients suffering from non-clonal cytopenias and dis-ease conditions other than MDS, in line with previousobservations (13,16,20); however, in such cases, anoverall lower number of milder alterations was detected,typically leading to lower IS (�1.5). Overall, theseresults are in line with those previously reported in theliterature in which the analysis of BM cells was re-stricted to CD34þ cells and/or the major compartmentsof both neutrophil and monocytic precursors (13,16),suggesting that immunophenotyping could improve thediagnostic efficiency of other conventional diagnosticcriteria (38). In line with previous studies (40), ourresults confirm that MDS recurrently show an alteredgranularity (SSC) for maturing neutrophils (11) andmonocytic cells (13), together with abnormal expressionof CD45 (18), CD64 (16), CD56 (13), CD11b/CD13 (20)and asynchronous shift to the left (18) among maturing

neutrophils as well as altered expression of CD13 (16),CD36, CD2 and CD56 on monocytic cells(13,16,19,20,39).

Interestingly, our results also showed a progressiveincrease in the number and severity of the immunophe-notypic abnormalities detected -as reflected by anincreasing IS-, from low to high grade MDS. Amongother parameters, those appearing to be increasinglyaltered among high vs low grade patients included thenumber of CD34þ HPC and CD34�/CD117þ precursorsdisplaying an aberrant phenotype; in contrast, the num-ber of CD34�/CD117þ precursor cells showing matura-tion commitment towards the neutrophil and/orerythroid lineages, progressively decreased. Such pro-gressive expansion of immature CD34þ and CD34�/CD117þ aberrant cells was also associated withdecreased percentages of CD34� B-cell precursors andboth CD34� maturing neutrophils and monocytes,which appeared to be blocked at intermediate stages ofmaturation. In contrast, increased numbers of NRBCwere detected only among low-risk MDS (RCMD and5q� syndrome; P ¼ 0.01), whereas mature B-lympho-cytes, basophils, mast cells and pDC remained at normallevels. Altogether, these findings are in agreement withthose previously reported on MDS by others and ourown group, based on both morphologic (15) and immu-nophenotypic (40) analyses regarding the phenotypicprofiles of different major compartments of BM cells andCD34þ HPC. Interestingly, in these studies an increasein the more immature compartment of CD34þ HPC isassociated with decreased numbers of CD34þ B-, neutro-phil-, pDC-, basophil- and erythroid-lineage precursorcells, except for low-grade MDS cases, where a shifttowards production of CD34þ neutrophil and erythroidlineage cells (both essential for patient survival), wasdetected at the expense of B-cell and pDC precursors.

Of note, among low-grade MDS, very similar profilesand IS were found in RA versus RCMD, suggesting theexistence of multilineage involvement in both patientgroups. In line with this, previous studies have alsoshown occurrence of inappropriate expression oflymphoid-associated markers among immature myeloidprecursors at similar frequencies, in RA and RCMDpatients (20). Altogether, these results suggest thatoccurrence of phenotypic aberrancies among maturingmyelomonocytic cells, might not always translate intoclearly altered morphologic features and that, the use ofimmunophenotyping in addition to cytomorphology,may contribute to a more robust discrimination betweenRCMD and pure RA with unilineage dysplasia, as alsopointed out by other groups (20,40).

Despite the fact that increasingly pronounced matura-tional defects are detected among the earliest compart-ments of CD34þ BM precursors from high- vs low- gradeMDS (25), these might also involve the CD34� compart-ments of maturing neutrophil and monocytic cells(13,16). In line with this, CD34� maturing neutrophilsfrom most patients with MDS showed a trend to retainan abnormally immature, altered phenotype.

PHENOTYPE IN MDS AND SURVIVAL 165

Cytometry Part B: Clinical Cytometry

Accordingly, a significant maturational blockade wasdetected once the CD11b and CD13 antigens weresimultaneously assessed (18) with accumulation of stageI and II CD11b�/CD34� neutrophil precursors(CD11b�/CD13hi myeloblasts and CD11b�/CD13lo pro-myelocytes) at the expense of CD11bþ/CD13lo-hi (stagesIII and IV) maturing neutrophils (myelocytes, metamye-locytes, bands and neutrophils); this was associated withdecreased numbers of CD16hi maturing neutrophils, asignificantly lower reactivity for early neutrophil-associ-ated markers (e.g., CD15, CD65 and CyMPO) togetherwith dysregulated CD45 expression, marked hypogranu-larity (low SSC) and aberrant expression of CD14 and/orCD56. A similar maturational arrest with increased num-bers of cells showing immature/altered phenotypes -decreased expression of monocytic-associated antigens(e.g., IREM-2, CD13, CD14 and CD64) and aberrantexpression of CD56 and/or CD2- was also observedamong maturing monocytic cells. Although several ofthese abnormalities have been previously described tobe associated with MDS (40), others (e.g., IREM-2 andCD2) are reported here for the first time.

Asynchronous expression of CD71 versus CD235a hasbeen previously reported on NRBC of around 80% of allpatients with MDS (11) in association with a greater pro-portion of immature red cells (11,19,26). In the presentstudy, we confirmed the existence of an altered expres-sion of both CD71 and CD235a in patients with MDS; inaddition, increased numbers of CD36�/lo NRBC werefound in up to 71% of all patients including both low-and high-grade MDS cases; overall these results supportthe notion (22) that the use of this antigen couldimprove the identification of altered erythroid pheno-types particularly among patients with low-grade MDS.The potential impact of time on the patterns of antigenexpression (e.g., CD11b expression on maturing neutro-phils) for EDTA-anticoagulated BM samples was specifi-cally excluded for freshly collected BM specimensprocessed within the first 24 hours after they wereobtained (data not shown).

Overall, our findings confirm the existence of a vari-able degree of impairment of BM hematopoiesis inpatients with low- versus high-grade MDS leading to aprogressively lower ability of MDS BM for multilineagematuration (25). In line with this, a clear associationwas found between the number and severity of thealtered phenotypes -IS- and the number of PB cytope-nias, the frequency of thrombocytopenic - but not ane-mic or leukopenic- patients and the proportion of caseswith high BM blast cell counts, in addition to both theWHO and IPSS subtypes of MDS. Interestingly, despitethe high number of phenotypic variables here analyzed(n ¼ 83), only a few showed a significant impact onpatient survival. These included the overall number ofCD34þ cells, the presence of aberrant CD34�/CD117þ

precursors, decreased numbers of CD34� mature neu-trophils and erythroid precursors, and increased num-bers of CD36�/lo NRBC. Thus, these variables couldrepresent the prognostically most informative pheno-

typic parameters which would reflect accumulation ofearly altered hematopoietic precursors in associationwith an impaired production of both mature erythroidcells and neutrophils, which are well-established prog-nostic factors (42,43). In line with this, increasinglyaltered phenotypes (e.g., expression of lymphoid-associ-ated markers) of immature myeloid precursors from RAand RCMD patients have also been associated with trans-fusion dependency and progression to advanced disease(20).

So far, several immunophenotypic scoring systemshave been proposed in which additional weight was ar-bitrary given to specific alterations which are thought tobe more clearly associated with MDS or high-grade MDS,without exploring the prognostic impact of the individ-ual parameters analyzed (13,23). Here, although individ-ual abnormalities had the same weight in the IS, inthose MDS cases with deep maturational impairment,overweighted score values may be obtained because ofredundant or interdependent alterations—e.g., caseswith notable increase of early precursors translating intodecreased mature cells. Indeed, in three intermediate-2and high risk MDS patients redundant abnormalitieswere observed (increased neutrophil stages I and II asso-ciated with decreased stages III and IV; data not shown),which added 8 points to an overall IS of 23, 27.5 and33, respectively. Further refinement of the IS may berequired to avoid redundancy. Despite the associationsdescribed above, multivariate analysis showed that theIS (low versus intermediate/high score), together withthe presence of thrombocytopenia and/or anemia, wasthe best combination of independent prognostic factorsfor predicting overall survival in MDS, the former vari-able showing the strongest impact on patient outcome.These results support and extend on previous observa-tions in patients with MDS studied at diagnosis (20) orbefore an allogeneic transplant (13), suggesting that thenumber and degree of the phenotypic abnormalitiesdetected could provide prognostic information comple-mentary to that of other conventional prognostic factors(e.g., IPSS). However, due to the relatively limited num-ber of cases analyzed, further studies in larger series ofpatients with a longer follow-up are required to confirmour preliminary observations.

In summary, in the present study we show systematicoccurrence of altered phenotypes in MDS; although sev-eral of these abnormalities were not specific of MDS andcould also be found in other nonclonal cytopenic condi-tions, they allow for an efficient discrimination betweenMDS and both normal and reactive BM, once the num-ber and degree of severity of the abnormalities detectedare simultaneously considered. Interestingly, progres-sively higher IS were found among MDS patients show-ing adverse prognostic features of the disease, theoverall degree of phenotypic alterations detected repre-senting a powerful independent prognostic factor foroverall survival. However, because of the high numberof immunophenotypic variables considered in this ISand the need to establish direct comparisons with

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normal BM to assign individual scores, the introductionof new analytical tools such as those proposed by theEuroFlow group (44,45) is urgently required for its rou-tine application in diagnosis and prognostic stratificationof patients with MDS. In addition, further multicentricstudies in larger series of patients with long follow-upare required to confirm our observations.

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