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Leukemia Research 36 (2012) 163– 168

Contents lists available at SciVerse ScienceDirect

Leukemia Research

jo ur nal homep age: www.elsev ier .com/ locate / leukres

uantity of clonal cells detected by conventional cytogenetic analysis correlatesith bone marrow blasts and survival in myelodysplastic syndromes

iyoung Kima, Soie Chunga, Cha Ja Seea, Sung-Soo Yoonb, Byoung Kook Kimb, Hyun Kyung Kima,ong Soon Leea,c,d,∗

Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, South KoreaDepartment of Internal Medicine, Seoul National University College of Medicine, Seoul, South KoreaDepartment of Tumor Biology, Seoul National University College of Medicine, Seoul, South KoreaCancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea

r t i c l e i n f o

rticle history:eceived 7 April 2011eceived in revised form 22 August 2011ccepted 23 August 2011vailable online 13 September 2011

a b s t r a c t

We performed quantitative and qualitative analyses of conventional cytogenetic analysis and interphaseFISH results in 87 MDS patients. The quantity of clonal cells for each chromosome of CCA did not correlatewith the result of iFISH (r, range 0.0761–1.0577). The clonal cell percentage in CCA was higher in patientswith >5% bone marrow blasts than those with <5% (44.7% vs. 23.1%, p = 0.017). Multivariate analysisshowed that a high quantity of clonal cells in CCA analysis is an independent prognostic factor for overall

eywords:uantitylonalityonventional cytogenetic analysis

survival in MDS (p = 0.012).© 2011 Elsevier Ltd. All rights reserved.

ISHDS

. Introduction

In practice, chromosomal abnormalities not only confirm thelonality of a disease but also predict the likelihood of progres-ion into acute leukemia and survival in myelodysplastic syndromeMDS) [1–6]. The presence of chromosomal abnormalities hasurned out to be one of the most important prognostic factors for

DS and has been incorporated into the International Prognosticcoring System (IPSS), along with the percentage of bone mar-ow blasts and the number of lineages involved in cytopenia [1–8].solated −Y, −5/5q−, −20q, and normal karyotypes imply a goodrognosis, while −7/7q− or complex abnormalities (abnormalities

n more than 3 types of chromosomes) suggest a poor prognosis5,6,8]. Recent studies by the German–Austrian MDS Study Groupnd the Spanish Group have analyzed the prognostic significance ofncommon abnormalities, including 1q gain, trisomy 8, and 12p−8,9].

Conventional cytogenetic analysis (CCA) and fluorescence in situybridization (FISH) are the most commonly used techniques toetect clonal changes in MDS [1,2,10–12]. FISH, a molecular cyto-

∗ Corresponding author at: Department of Laboratory Medicine, Seoul Nationalniversity College of Medicine, 101 Daehang-ro, Jongno-gu, Seoul 110-744, Southorea. Tel.: +82 2 2072 3986; fax: +82 2 747 0359.

E-mail address: soonlee@plaza.snu.ac.kr (D.S. Lee).

145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2011.08.021

genetic method, has several advantages over CCA. FISH can detectsubmicroscopic abnormalities owing to its sensitivity [12]. BothCCA and FISH can provide information about clone size since theycan quantitate the clonal cells [12]. However, in contrast to CCA,interphase FISH (iFISH) does not require viable cells and can be car-ried out with non-dividing cells. Nevertheless, CCA is the standardtechnique recommended by the IPSS algorithm, as it enables thedetection of complex abnormalities that could be missed in FISH[13].

Most studies have investigated clonal chromosomal abnormal-ities in MDS by focusing on the relationship between the presenceor absence of an abnormality of a certain chromosome and theprognosis of MDS [1,2,8–12], but not on the quantity of leukemiccells burdened with CCA. Little is known about the utility of thequantitative results of CCA and iFISH in clinical practice. We inves-tigated the roles of CCA and FISH results in predicting the outcomeof MDS patients. The results of CCA and FISH were compared bothin qualitative and quantitative aspects. We analyzed the prognosticsignificance of the quantity of clonal cells in CCA or FISH in MDS.

2. Materials and methods

2.1. Patients

Data for the study was accumulated from 129 newly diagnosed, de novo MDSpatients of Seoul National University Hospital between April 2000 and March 2010.Due to suboptimal number of metaphase cells (less than 20), only 87 patients (54

164 M. Kim et al. / Leukemia Research 36 (2012) 163– 168

Table 1The incidence of clonal chromosomal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ in conventional cytogenetic analysis (CCA) and interphase fluorescencein situ hybridization (iFISH) in myelodysplastic syndromes.

−5/5q− −7/7q− +8 −20/20q− +1/1q

CCA+ 9 (10.3%) 7 (8.0%) 10 (11.5%) 5 (5.7%) 9 (10.3%)CCA+, iFISH− – – 1 (1.1%) 1 (1.1%) 1 (1.1%)

m1r1retltR

2

tbwst

2

8wcemaoots

2

IBtitLpnP

3

3ci

mcimh+p−i

iFISH+ 9 (10.3%) 9 (10.3%)

CCA−, iFISH+ – 2 (2.3%)

CCA+ and/or iFISH+ 9 (10.3%) 9 (10.3%)

en and 33 women) were included from the analysis. The patients’ age was between9.6 and 83.0 years (mean = 57.6 years). The patient group included 25 cases ofefractory anemia (RA), 5 cases of refractory anemia with ringed sideroblasts (RARS),1 cases of refractory cytopenia with multilineage dysplasia (RCMD), 24 cases ofefractory anemia with excess blasts-1 (RAEB-1), 19 cases of refractory anemia withxcess blasts-2 (RAEB-2), and 3 cases of unclassifiable MDS (MDS-U), according tohe WHO 2001 classification. Data concerning patient deaths and acute myeloideukemia (AML) progression, obtained in May 2010, showed that 15 had progressedo AML, 21 died, and 66 survived. This study was approved by the Institutionalesearch Board of the Seoul National University Hospital (IRB No. H-1005-001-316).

.2. Conventional cytogenetic analysis

Cytogenetic studies using standard techniques were performed as a part ofhe diagnostic work-up. At least 20 metaphases were analyzed, whenever possi-le. Clonal abnormalities were defined as 2 or more cells with the same additionalhole chromosome or chromosome rearrangements, or 3 or more cells with the

ame chromosome missing. Chromosomal abnormalities were described accordingo the International System for Human Cytogenetic Nomenclature 2005 [14].

.3. Interphase fluorescence in situ hybridization

We used the iFISH technique to detect the abnormalities of chromosomes 5, 7,, 20, and 1 (−5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+). The following probesere used: LSI (locus-specific identifier) EGR1/D5S23, D5S721 Dual Color Probe for

hromosome 5q; LSI D7S522/CEP 7 Probe for chromosome 7q; CEP (centromerenumeration probe) 8 DNA Probe for chromosome 8; LSI D20S108 Probe for chro-osome 20q; and LSI p58 (1p36)/TelVysion 1p/LSI 1q25 Probe for chromosome 1;

ll probes were obtained from Vysis Inc. (Downers Grove, IL, USA). The normal cut-ff values of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ based on the mean (3 SD)f 40 negative controls, were 2.0%, 2.0%, 2.0%, 2.0%, and 1.5%, respectively. No lesshan 200 nuclei of a sample were scored for deletion and rearrangement in eachample. Results were described according to the ISCN (2005) criteria [14].

.4. Statistical analysis

All statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago,L, USA). Chi-square test was applied for the comparison of categorical variables.ivariate correlation analysis was used to evaluate the correlation between two con-inuous variables, and r (Pearson’s correlation coefficient) and r2 were consideredn the evaluation. Mann–Whitney U-test was applied for the comparison of con-inuous variables. Survival curves were estimated using the Kaplan–Meier method.og-rank tests were used to conduct univariate comparisons of overall survival androgression-free survival among subgroups. Multivariate analyses adjusted for sig-ificant prognostic factors were performed using Cox’s hazard regression model.rognostic significance was evaluated in terms of 95% confidence intervals.

. Results

.1. Clonal abnormalities of chromosomes 5, 7, 8, 20 and 1 inonventional cytogenetic analysis and interphase fluorescencen situ hybridization

Conventional cytogenetic analysis (CCA) detected clonal chro-osomal abnormalities of any chromosomes in 40 (46.0%), and

lonal abnormalities of −5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+n 31 (35.6%). Twelve (13.8%) had complex chromosomal abnor-

alities (≥3 abnormalities) in CCA. Interphase fluorescence in situybridization (iFISH) for −5/5q−, −7/7q−, +8, −20/20q−, and

1/1q+ detected clonal chromosomal abnormalities in 36 (41.4%)atients. The incidence of clonal chromosomal abnormalities of5/5q−, −7/7q−, +8, −20/20q−, and +1/1q+ observed in CCA and/or

FISH is summarized in Table 1. The clonal abnormalities were

14 (16.1%) 6 (6.9%) 9 (10.3%)5 (5.7%) 2 (2.3%) 1 (1.1%)

15 (17.2%) 7 (8.0%) 10 (11.5%)

found in 9 (10.3%), 9 (10.3%), 15 (17.2%), 7 (8.0%), and 10 (11.5%)of the subjects, respectively, by CCA and/or iFISH.

The discrepancies between CCA and iFISH were observed in12 (13.8%) patients, and their clinicopathological characteristicsare detailed in Table 2. CCA detected clonal abnormalities of +8,−20/20q−, and +1/1q+ in 1 (1.1%), 1 (1.1%), and 1 (1.1%) patientswho did not show those abnormalities in iFISH. The sizes of clonesdetected in CCA were variable (18.2–83.3%). iFISH detected sub-microscopic clonal abnormalities of −7/7q−, +8, −20/20q−, and+1/1q+ in 2 (2.3%), 5 (5.7%), 2 (2.3%), and 1 (11%) patients, and thesizes of clones in iFISH ranged from 5.0 to 84.0%. Among them, 6patients (010, 016, 019, 028, 046 and 067) showed normal kary-otype in CCA (12.8% of 47 patients with normal karyotype).

3.2. Prognostic significance of qualitative results of conventionalcytogenetic analysis and interphase fluorescence in situhybridization

Out of 12 with discrepancies between CCA and iFISH, the IPSSrisk group changed in 1 case (8.3%: patient 003) from Int-2 to Highdue to the clonal abnormality detected only by CCA, and in 3 cases(25.1%: patient 016, 028, and 046) from Int-1 to Int-2 due to thesubmicroscopic clonal abnormalities detected by iFISH. In the totalstudy population, the rate of evolution to AML, leukemia free sur-vival and overall survival of each IPSS risk group are as follows:0.0%, 654 days and 654 days in Low risk group; 10.2%, 617 daysand 627 days in Int-1 risk group; 33.3% 382 days and 515 days inInt-2 risk group; 22.2%, 275 days and 300 days in High risk group.Patient 016 and 028 showed similar survival duration to Int-1 riskgroup, while patient 003 and 046 showed similar survival durationto High risk group and Int-2 group, respectively. Statistical compar-ison of survival could not be performed due to too small number ofpatients (4) who changed IPSS group.

3.3. Qualitative analysis of clonal chromosomal abnormality

Clonal cells with chromosomal abnormalities were detected byCCA in 28.0%, 0.0%, 63.6%, 54.2%, 57.9% and 66.7% of the 25 RA,5 RARS, 11 RCMD, 24 RAEB-1, 19 RAEB-2, and 3 MDS-U patients,respectively. The incidence was similar in iFISH analysis: the pres-ence of any clonal abnormality of −5/5q−, −7/7q−, +8, −20/20q−,and +1/1q+ was detected in 32.0%, 20.0%, 45.5%, 45.8%, 42.1% and66.7% of RA, RARS, RCMD, RAEB-1, RAEB-2 and MDS-U patients,respectively. There was no association between the incidence ofclonal abnormality and the percentage of BM blasts, cytopenia, orMDS subtype (p > 0.05).

3.4. Quantitative analysis of clonal chromosomal abnormality

When only the patients with clonal abnormalities detectedby CCA and/or FISH are considered, the quantitative results ofclonal cells detected in the 2 methods yielded a variable degree of

agreement (poor to substantial, depending on the chromosomesinvolved): −5/5q−: r = 1.0577, r2 = 0.5022; −7/7q−: r = 0.0761,r2 = 0.0181; +8: r = 0.5164, r2 = 0.5179; −20/20q−: r = 0.6805,r2 = 0.8014; and +1/1q+: r = 0.2569, r2 = 0.1547 (Fig. 1). The

M. Kim et al. / Leukemia Resea

Tab

le

2C

lin

icop

ath

olog

ical

feat

ure

s,

con

ven

tion

al

cyto

gen

etic

anal

ysis

(CC

A)

resu

lts

and

inte

rph

ase

flu

ores

cen

ce

in

situ

hyb

rid

izat

ion

(iFI

SH)

resu

lts

of

12

pat

ien

ts

wit

h

dis

crep

anci

es

betw

een

CC

A

and

iFIS

H.

Cas

e

no.

(sex

/age

)H

b

(g/d

L)–W

BC

(/u

L)–A

NC

(/u

L)–P

LT(×

103/�

L)

Dia

gnos

isC

ellu

lari

ty(%

)K

aryo

typ

e

by

CC

AiF

ISH

IPSS

Gro

up

(Sco

re)

Eith

er

CC

A

or

FISH

→

CC

A

&FI

SH

Ove

rall

surv

ival

(day

s)

003

(M/2

5)

6.6–

1990

–199

4–12

4

RA

EB-2

50–6

0

47,X

Y,a

dd

(2)(

p25

),+m

ar[5

]/47

,idem

,du

p(1

q)[2

5]

No

abn

orm

alit

y

det

ecte

d

Int-

2

(2.0

) →

Hig

h

(3.0

)

601

0

(M/6

0)

4.5–

2830

–203

2–50

RA

RS

NA

a46

,XY

[20]

−20/

20q−

(27.

5%)

Int-

1

(0.5

) →

Int-

1

(0.5

) 10

1601

6

(F/6

1)

10.0

–117

0–24

6–85

RA

EB-1

20–3

0

46,X

X[2

0]

+8

(36.

0%)

Int-

1

(1.0

) →

Int-

2

(1.5

) 88

901

7

(M/8

3)8.

0–95

20–3

903–

57

RA

EB-2

NA

44,X

,−5,

i(8)

(q10

),d

er(8

;9)(

q10;

q10)

,i(13

)(q1

0),−

16,+

mar

[3]/

45,X

Y,id

em[1

6]/4

6,X

Y[1

]−5

/5q−

(81.

0%),

−7/7

q−(8

4.0%

),

+8

(5.3

%)

Hig

h

(3.0

) →

Hig

h

(3.0

)

162

019

(F/4

4)

11.1

–239

0–12

79–7

0

RA

20–3

0

46,X

X[2

0]

+8

(9.3

%)

Int-

1

(0.5

) →

Int-

1

(1.0

)

1592

028

(M/2

0)11

.1–1

640–

1032

–31

RA

20–3

0

46,X

Y[2

0]−7

/7q−

(31.

7%)

Int-

1

(0.5

) →

Int-

2

(1.5

)

1079

036

(F/7

7)

8.9–

5730

–154

7–13

0

RA

EB-1

80–9

0

45,X

X,d

el(5

)(q1

5q33

),−7

,der

(12)

t(7;

12)(

p11

.2;p

11.2

),d

el(1

8)(q

21)[

18]/

40–4

5,sl

,del

(7)(

q22)

,−16

,−17

,−20

,+1–

2

mar

[cp

4]−5

/5q−

(65.

0%),

−7/7

q−(5

9.0%

)In

t-2

(2.0

) →

Int-

2

(2.0

)

153

046

(M/7

0)10

–192

0–42

2–82

RA

EB-1

70–8

0

46,X

Y[2

0]+8

(11.

0%)

Int-

1 (1

.0) →

Int-

2

(1.5

)45

2

(434

b)

048

(M/7

2)6.

9–72

30–4

338–

224

RA

EB-1

90–1

00

47,X

Y,+

8[20

]

+8

(79.

5%),

+1/1

q

(5.0

%)

Int-

1

(1.0

) →

Int-

1

(1.0

)

511

065

(F/6

8)6.

2–11

00–2

31–3

RA

EB-2

80–9

0

46,X

X,d

el(5

)(q1

3q33

),in

v(9)

(p11

q13)

,−13

,der

(18)

t(13

;18)

(q14

;q23

),+

add

(?22

)(q?

13),

0–11

dm

in[5

]/45

–55,

sl,+

X,+

1,+8

,+11

[cp

7]/

46,s

l,ad

d(1

6)(q

?24)

[4]/

46,X

X,in

v(9)

(p11

q13)

[4]

−5/5

q

(78.

5%),

+1/1

q(3

5.5%

)H

igh

(2.5

) →

Hig

h

(3.0

)57

067

(M/7

2)

7.7–

3170

–139

5–15

6

RA

EB-1

70–8

0

46,X

Y

[20]

−20/

20q−

(15.

0%)

Int-

1

(1.0

) →

Int-

1

(1.0

)

429

082

(M/7

5)8.

3–19

80–5

30–1

9R

AEB

-180

–90

39–4

5,X

Y,+

del

(3)(

q10)

,der

(?5)

t(?5

;?12

)(q?

15;q

13)?

del

(5(p

?),−

7,−2

1[1]

/39–

42,X

,sl,−

Y,d

el(3

)(p

23),

+ad

d(9

)(q?

10),−1

2,−1

5,−1

7,−

22[1

3]/4

0–45

,sl,a

dd

(19)

(q13

.1),

+2m

ar[6

]/46

,XY

[3]

−5/5

q−(4

0.0%

),

−7/7

q−(2

0.0%

),

+8

(16.

0%)

Int-

2

(2.0

) →

Int-

2

(2.0

)18

4

aN

ot

avai

labl

e

du

e

to

inad

equ

ate

qual

ity

of

BM

biop

sy

sect

ion

.b

Leu

kem

ia

free

surv

ival

du

rati

on

in

the

case

evol

ved

to

AM

L.

rch 36 (2012) 163– 168 165

quantity of clonal cells from total metaphase cells in CCA was higherin the 43 patients with >5% BM blasts than in the 44 patients with<5% BM blasts in CCA (44.7% vs. 23.1%, p = 0.017; Fig. 2). The differ-ence in the quantity of clonal cells in CCA between patients with>10% and those with 5–10% blasts was not statistically significant(p > 0.05). However, the quantity of clonal cells from the total inter-phase cells in iFISH was independent of the percentage of BM blasts:44 patients had <5% BM blasts and 43 patients had >5% BM blasts(18.1% vs. 24.0%, p > 0.05). The difference in the quantity of clonalcells in iFISH between patients with >10% and those with 5–10%blasts also showed no statistically significance (p > 0.05).

3.5. Prognostic significance of quantitative results ofconventional cytogenetic analysis and interphase fluorescencein situ hybridization

In univariate analysis, the quantity of clonal cells in CCA > 50.0%,the presence of clonal cells in iFISH, the quantity of clonal cells iniFISH > 50.0%, isolated abnormalities of −5/5q− (detected by CCAand/or FISH), and the percentage of BM blasts >5% showed prog-nostic significance for overall survival (p < 0.05). In multivariateanalysis, the quantity of clonal cells in CCA > 50.0% (p = 0.012) wasthe only independent prognostic factor for overall survival in thisstudy population (Fig. 2).

The following factors showed prognostic significance for theprogression-free survival into AML cases in univariate analysis(p < 0.05): the quantity of clonal cells in CCA > 50.0%, the quantity ofclonal cells in iFISH > 50.0%, isolated abnormalities of −5/5q− and+8, and the percentage of BM blasts >5%. In multivariate analysis,the percentage of BM blasts was the only independent prognos-tic factors for the progression-free survival into AML in this studygroup (p < 0.001). Individual cytogenetic abnormalities were notconsidered in the adjustment.

4. Discussion

Little is known about the clinico-biological significance of thequantity of clonal cells in MDS. We performed both qualitative andquantitative analyses of the results of CCA and iFISH in MDS, andinvestigated their clinical significance.

Qualitative analysis of CCA and iFISH data revealed a 13.8%(12/87) incidence of discrepancies between CCA and iFISH. Thisincidence could not be compared with others since too few studieshave been performed. Among those classified with normal kary-otypes by CCA, 12.8% (6/47) showed clonal abnormalities in iFISH,and they showed cytopenia, dysplasia and/or increased BM blasts astypical MDS does. Differences in the sensitivities of the two meth-ods might have caused the discrepancy. Except for 1 case (patient017, −7/7q−, 84%), the sizes of clones detected only by iFISH wererelatively small (5.3–36.0%). It is in the same context with the pre-vious studies with normal karyotype MDS which showed that theproportion of cells with submicroscopic abnormalities detectedonly in iFISH reached at most 30% [1,2,11]. The low mitotic indexof the abnormal clone could be an explanation for the discrepancy.The clone in MDS is not limited to the blast, but is also present in thematuring trilineage cells, therefore the dividing ability of the clonemust be variable. Another explanation could be that only a subsetof clone is involved in chromosomal abnormalities. The previousstudies with normal karyotype MDS suggested the subset of clonewith chromosomal abnormalities are related to the disease pro-gression as the size of clone in iFISH correlated with BM blast%, and

it increases as the disease progressed to acute leukemia [1,2,11].Further study is required to clarify this issue since the correlationwith BM blast% and the quantity of clonal cells in iFISH were poorin our study with MDS with normal and abnormal karyotype.

166 M. Kim et al. / Leukemia Research 36 (2012) 163– 168

F tic anp f clona

wpibNp0si

ig. 1. Correlation of the proportion of clonal cells between conventional cytogenelastic syndromes. x, the proportion of clonal cells in iFISH; and y, the proportion o

The IPSS risk grouping was changed only in a subset of patientsith discrepancies (33.3%, 4/12), due to the fact that the remainingatients already presented a complex karyotype in CCA prior to

FISH analysis. Thus, the statistical significance of IPSS risk groupingefore and after considering iFISH results could not be evaluated.evertheless, the combination of CCA and iFISH is beneficial for

redicting the outcome for some MDS patients as shown in case03 and 046 who have shorter overall survival and leukemia freeurvival estimated by IPSS determined only either by CCA or byFISH.

alysis (CCA) and interphase fluorescence in situ hybridization (iFISH) in myelodys-l cells in CCA. (a) −5/5q−; (b) −7/7q−; (c) +8; (d) −20/20q−; and (e) +1/1q.

The data from the quantitative analysis of CCA and iFISH in thepresent study is summarized as follows: (i) the quantity of clonalcells in CCA and iFISH did not correlate well with each other (r,range 0.0761–1.0577); (ii) the percentage of BM blasts correlatedwith the quantity of clonal cells in CCA (p = 0.017) but not with thequantity of clonal cells in iFISH (p > 0.05); (iii) the high quantity

of clonal cells in CCA was a poor prognostic factor in predictingthe survival of MDS patients either independently (overall survival,p = 0.012) or in relation to BM blast% (leukemia-free survival). Tothe best of our knowledge, no study has reported these findings.

M. Kim et al. / Leukemia Research 36 (2012) 163– 168 167

Fig. 2. Prognostic significance of the proportion of clonal cells in conventional cytogenetic analysis (CCA) in myelodysplastic syndromes. (a) Correlation between thep correb

TtitCCbdtiLt9w

a

roportion of clonal cells in CCA and the percentage of bone marrow blasts; and (b)etween the proportion of clonal cells in CCA leukemia-free survival.

he differences we observed could be attributed to the differentypes of cells used for the 2 techniques, metaphase cells for CCA andnterphase cells for iFISH. Cells that are viable and able to proliferateend to form metaphase cells more easily than quiescent cells inCA; thus, the high percentage of blasts might have affected theCA results. In contrast, the results of iFISH might be less affectedy the percentage of blasts since the technique is performed on non-ividing, interphase cells. It has been suggested that the increase inhe size of abnormal clones, which was observed by iFISH, happensn all patients undergoing leukemic transformation [1]. However,i et al. showed that almost all cases of MDS had higher clonal cellshan blast quantity, and that some RA patients showed more than

0% clonal cells [15]. Our unpublished observations are concordantith the latter.

It has been demonstrated that the presence of clonal cells is poor prognostic factor in MDS [1,2]. Bernasconi et al. [2] and

lation between the proportion of clonal cells in CCA overall survival; (c) correlation

Rigolin et al. [1] showed that MDS patients with a normal kary-otype in CCA but with submicroscopic abnormalities in FISH hadeither a short disease progression-free survival or overall survival.The identity of the chromosomes involved in the clonal change wasneither considered by these 2 studies nor in our study. A notablefinding of our study is that a high quantity of clonal cells in CCAwas an independent prognostic factor in predicting a short overallsurvival (p = 0.012), and it affects leukemia-free survival as well, inrelation to BM blast%.

Our study confirmed that CCA and iFISH analysis are com-plementary to each other by detecting cytogenetic abnormalitiesoverlooked in either method. Quantitative analyses of CCA and

iFISH results showed that the quantity of clonal cells in CCA andin iFISH do not correlate with each other, and that the high quan-tity of clonal cells in CCA could be used as a surrogate marker inpredicting survival in MDS patients.

1 Resea

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cknowledgements

This work was supported in part by (i) the Korea Science andngineering Foundation (KOSEF) funded by the Ministry of Educa-ion, Science, and Technology (20100020584). Tae Young Kim andora Oh are grateful for being awarded a BK21 fellowship, (ii) arant (10172KFDA993) from Korea Food & Drug Administration in010, and (iii) Basic Science Research Program through the Nationalesearch Foundation of Korea (NRF) Funded by the Ministry of Edu-ation, Science and Technology (2010-0013651).

Contributions. M.K. wrote the draft, M.K. and S.C. analyzed data,.J.S. performed the cytogenetic analysis, S.S.Y. and B.K.K. clini-ally treated the MDS patients, H.K.K. and D.S.L. diagnosed the MDSatients, and D.S.L. designed the study, supervised the cytogeneticnalysis, and reviewed the draft.

Conflict of interest statement. Authors do not have any conflictsf interest to disclose.

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