Classical and molecular cytogenetic abnormalities and outcomeof childhood acute myeloid leukaemia: report from a referralcentre in Israel

Extensive cytogenetic analyses of the leukaemias have demon-

strated an association of recurrent chromosomal aberrations

with the clinical and biological diversity of acute myeloid

leukaemia (AML) and provided insight into the genetic

changes that underlie leukaemogenesis, thereby opening new

horizons for profiles of microarray gene expression pathways

and targeted treatment strategies (Rowley, 2000, 2001; Will-

man, 2001; Haferlach et al, 2003; Yagi et al, 2003). A few of the

more common specific cytogenetic/molecular abnormalities,

namely, t(8;21)/AML1-ETO, t(15;17)/PML-RARA, inv(16)/

t(16;16)/CBFb-MYH11 and t(11q23)/MLL, have recently been

recognized by the World Health Organization (WHO) classi-

fication as distinct disease categories, associated with the

French-American-British (FAB) groups (Harris et al, 1999;

Vardiman et al, 2002).

The strong association of diagnostic karyotype with out-

come, demonstrated in some large adult studies, has rendered

cytogenetics the most valuable prognostic factor for treatment

selection (Bloomfield et al, 1997; Grimwade et al, 1998;

Grimwade, 2001; Mrozek et al, 2001). However, for some less

frequent non-random aberrations, the cytogenetic prognostic

classification is still inconsistent owing to the small number of

patients and the variable treatment modalities used in different

study groups (Grimwade et al, 1998, 2002; Slovak et al, 2000;

Byrd et al, 2002).

The reported childhood AML series consist of a relatively

smaller number of patients and show a different distribution of

cytogenetic subsets (Woods et al, 2001) and sometimes a

different response to treatment from adults (Martinez-Climent

et al, 1995a; Grimwade et al, 1998; Raimondi et al, 1999;

Batia Stark,1,2 Marta Jeison,2

Leticia Glazer Gabay,2 Jacques

Mardoukh,2 Drorit Luria,3 Irit Bar-Am,4

Gali Avrahami,1 Yossef Kapeliushnik,5

Dalia Sthoeger,6 Gavriel Herzel,7

David M. Steinberg,8 Ian J. Cohen,1

Yacov Goshen,1 Jerry Stein,1

Rina Zaizov1 and Isaac Yaniv1

1Centre of Pediatric Hematology/Oncology,2Cancer Cytogenetic Laboratory and 3Flow

Cytometry Unit, Schneider Children’s Medical

Centre of Israel, Petah Tiqva, and Sackler Faculty

of Medicine, Tel Aviv University, Tel Aviv,4Applied Spectral Imaging, Ltd, Migdal Ha’emek,5Soroka Hospital, Beer Sheva, 6Kaplan Hospital,

Rehovot, 7Haemek Hospital, Afula, and8Department of Statistics and O.R., Tel Aviv

University, Tel Aviv, Israel

Received 5 January 2004; accepted for

publication 15 April 2004

Correspondence: B. Stark, The Center of

Pediatric Hematology/Oncology and the Cancer

Cytogenetic Laboratory, Schneider Children’s

Medical Centre of Israel, Petah Tiqva 49202,

Israel. E-mail: bstark@clalit.org.il

Summary

The incidence of cytogenetic abnormalities in childhood de novo acute

myeloid leukaemia (AML) and its prognostic significance was assessed in an

Israeli paediatric referral centre. Cytogenetic analysis was successful in 86 of

97 children (<20 years of age) diagnosed between 1988 and 2002 with de

novo AML. Fluorescence in situ hybridization analysis detected new

information in 11 of them, leading to reassignment in cytogenetic group

classification. The incidence of the various cytogenetic subgroups was as

follows: normal – 9%; t(11q23) – 22%; t(8;21) – 13%; t(15;17) – 8%;

inv(16) – 3Æ4%; abn(3q) – 4Æ6%; 7/7q-(sole or main) – 5Æ8%; del(9q)(sole)

and +21(sole) – 4Æ6% each; t(8;16) – 2Æ3%; t(6;9), t(1;22), +8(sole) – 1Æ1%each; and miscellaneous – 18%. The overall survival (OS) and event-free

survival (EFS) (4 years) for 94 patients treated with the modified Berlin-

Frankfurt-Munster (BFM) AML protocols (non-irradiated) were 59Æ9%(SE ¼ 5%) and 55Æ7% (SE ¼ 5%), respectively, and for the favourable

t(8;21), t(15;17) and inv(16), OS was 60% (SE ¼ 15%), 83% (SE ¼ 15%)

and 100% respectively. For the normal group it was 62% (SE ¼ 17%),

miscellaneous 64% (SE ¼ 12%), t(11q23) 44Æ6% (SE ¼ 11%) and of the

)7/7q-, del(9q)(sole) or t(6;9), none had survived at 4 years. The incidence

of cytogenetic subgroups in the Israeli childhood AML population and their

outcome were similar to other recently reported paediatric series.

Cytogenetic abnormalities still carry clinical relevance for treatment

stratification in the context of modern chemotherapy.

Keywords: cytogenetics, childhood acute myeloid leukaemia.

research paper

doi:10.1111/j.1365-2141.2004.05038.x ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337

Woods et al, 2001; Wells et al, 2002; Forestier et al, 2003;

Harrison et al, 2003). Moreover, geographic differences in the

frequency of cytogenetic subsets have been noted in adult and

childhood AML (Johansson et al, 1991; Biondi et al, 1994;

Nakase et al, 2000).

The aim of this study was to assess the demography of

cytogenetic abnormalities and their correlation with clinical

and biological features and impact on outcome in children

with de novo AML, who were diagnosed at a referral centre in

Israel and treated with the Berlin-Frankfurt-Munster (BFM)-

based AML protocols (Creutzig et al, 1999, 2001). Fluores-

cence in situ hybridization (FISH) analysis, using a panel of

commercial AML probes, was retrospectively performed on all

available, adequate-quality cytogenetic bone marrow (BM)

pellets to confirm and complement the information gained

from conventional cytogenetics.

Patients and methods

Patients

The study sample consisted of 97 newly diagnosed de novo

AML patients younger than 20 years, who were diagnosed at

the Schneider Children’s Medical Centre of Israel (88 patients),

Soroka Hospital (six patients), and Kaplan Hospital (three

patients) between July 1988 and January 2003. Four children

with Down syndrome and one with an undefined constitu-

tional non-Fanconi birth defect were included.

Treatment

Treatment for 94 patients (excluding another three patients

treated elsewhere) was based on the AML-BFM-87 (July 1988–

July 1996; 52 patients), AML-BFM-93 (August 1996–July 1998;

20 patients), and AML-BFM-98 (August 1998–January 2003;

22 patients) protocols (Creutzig et al, 1999, 2001). Modifica-

tions included mainly substitution of preventive cranial

irradiation with additional five intrathecal triple (TIT) cytara-

bine (Ara-C) methotrexate (MTX), and hydrocortisone injec-

tions during maintenance. Systemic chemotherapy was

administered without randomization, and all the patients

received the higher risk protocol. In the AML-BFM-87

protocol, patients who completed the second intensification

course were eligible for allogeneic bone marrow transplanta-

tion (BMT) from a human leucocyte antigen (HLA)-matched

sibling, if available, conditioning with cyclophosphamide and

busulphan (actually performed in three patients), or for

autologous BMT with conditioning with melphalan 180 mg/m2

(13 patients), or for continuation with maintenance chemo-

therapy for 1 year (29 patients). In the BFM-93 and -98

protocols, high-risk patients only were offered allogeneic BMT

from an HLA-matched sibling (actually performed in 11

patients). Patients in the non-high-risk group were eligible for

autologous BMT (performed in 14 patients) or maintenance

chemotherapy (nine patients). The non-high risk group was

defined similarly to the AML-BFM criteria as M3/t(15;17),

t(8;21), or M1/2 with Auer rods, inv(16) or M4 with

eosinophilia (Creutzig et al, 1999).

Diagnosis

The diagnosis and FAB subtypes of AML, with or without

evidence of dysplasia or prior myelodysplastic syndrome (MDS)

(Bennett et al, 1985a; Harris et al, 1999; Vardiman et al, 2002),

was determined by Wright-Giemsa-stained BM smears and

cytochemical staining with myeloperoxidase, Sudan-black,

a-naphthyl acetate esterase with and without sodium fluoride

inhibition, chloroacetate-esterase, periodic acid-Schiff, and acid

phosphatase. The diagnosis of M0, M6 and M7 subtypes

required confirmation of immunophenotype and/or electron

microscopywithplatelet peroxidase staining (Bennett et al, 1985b).

Immunophenotyping of the BM samples was performed by

flow cytometry using a FACScan (Becton Dickinson, San Jose,

CA, USA). The monoclonal antibodies tested were for the

panmyeloid antigens CD13 and CD33; myeloid granulocyte

marker CD15; monocytic macrophage marker CD14; erythroid

marker glycophorin A (GpA); megakaryocyte platelet markers

CD41 (GPIIb), CD42 (GPIIb), and CD61 (platelet GPIIIa);

non-lineage restricted marker CD11c; precursor markers

CD34, HLA-DR; anticytoplasmatic myeloperoxidase Cy

MPO; B-cell markers CD19 and CD10; T-cell markers CyCD3,

CD2, CD5, CD7, CD4; natural killer (NK) cell: CD56.

Cytogenetics

Cytogenetic analysis was performed on metaphase cells from

direct or short-term unstimulated BM cultures using the

trypsin-Giemsa banding technique. Chromosomal abnormal-

ities were identified and classified according to the Interna-

tional System for Human Cytogenetic Nomenclature (ISCN)

(Mitelman, 1995). Patients were classified as having a normal

karyotype after 20 normal metaphases were analysed, except for

two patients in whom only 12 and 18 metaphases were available.

Molecular-cytogenetic analysis

Since 1998, interphase FISH analysis with a panel of com-

mercially available probes has been used for the diagnostic

work-up of all new AML patients complementary to conven-

tional cytogenetics. In addition, retrospective FISH analysis

was performed on cytogenetic pellets of adequate quality from

patients treated before 1998. The AML panel included

commercially available probes for the detection of t(11q23),

t(8;21), t(15;17), inv(16), t(9;22), deletion 5/5q, deletion 7/7q,

del(20q), and extra chromosome 8. Most of the panel was used

when new or additional information was expected: if cyto-

genetic results were inadequate (i.e. non-dividing cells, non-

assessable metaphases, or normal), or non-specific chromo-

somal changes or complex karyotypes were demonstrated, or

in the presence of known additional secondary changes.

Cytogenetics, Prognosis in Childhood Acute Myeloid Leukaemia

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337 321

Otherwise, FISH was applied with a selected probe only to

confirm a suspected specific cytogenetic AML translocation/

aberration or a specific AML FAB morphological subtype.

FISH probes. MLL-dual-colour break-apart, AML1/ETO dual-

colour dual fusion translocation, PML/RARA dual-colour

translocation, CBFb dual-colour break-apart, BCR/ABL dual-

colour extra signal, LSI EGR1 (5q31)(200 kb)/D5S721 D5S23

(5p15Æ2) (450 kb) for 5q31 deletion. LSI D7S486(7q31)

(200 kb)/CEP 7 for 7q31 deletion. D20S108 single colour for

deletion in 20q12 locus, LSI21q22Æ13-q22Æ2 (200 kb) and CEP 8.

All probes were from Vysis Inc. (Downers Grove, IL, USA).

Dual-colour FISH was performed according to the manu-

facturer’s protocol, as previously described (Stark et al, 2002).

FISH signals were visualized with an Olympus BX50 fluores-

cent microscope (Olympus Opticals, Tokyo, Japan), and

images were captured with the Cytovision System (Applied

Imaging, Newcastle-upon-Tyne, UK).

In the majority of cases, 200 interphase nuclei were screened,

but in a few cases lower number of nuclei were available. The

cut-off level for the diagnosis of deletion and trisomies, was set

at the mean percentage of false positive results + 3 SD, i.e.

9Æ5% for 7q-, 8Æ3% for 5q-, 8Æ5% for +21.

Spectral karyotype analysis

Slides were hybridized with the spectral karyotype (SKY) probe

mixture obtained from Applied Spectral Imaging (Migdal

Ha’emek, Israel) as previously described (Stark et al, 2000).

The SKY slides were analysed with the skyview software on

the SD300 Spectral Imaging system (Applied Spectral Ima-

ging).

Cytogenetic/molecular cytogenetic hierarchical classification.

Patients were categorized by the cytogenetic group that

reflected the recognized primary abnormality. In this manner,

each patient was counted only once. The cytogenetic categories

were as follows: normal karyotype, known recurrent t(8;21),

t(15;17), inv(16), t(11q23), 3q abnormality, t(6;9), t(8;16) and

variant, t(1;22), monosomy 7(sole)/del(7q), del(9q)(sole),

trisomy 8(sole), trisomy 21(sole), and miscellaneous clonal

aberrations. Monosomy 7, del(9q), +8, +21, were considered

primary when they appeared cytogenetically as the sole

aberration confirmed by FISH, ruling out any other primary

abnormality. del(7q), when appearing cytogentically in a

complex karyotytpe in all metaphases and confirmed by

FISH, was also considered primary. These abnormalities were

considered secondary when they appeared cytogenetically in

addition to other known primary translocations and/or were

detected by FISH only in a subpopulation of cells. The

cytogenetic group was usually determined by conventional

karyotyping and rarely by SKY. When FISH analysis disclosed

an AML-specific rearrangement, namely MLL splitting, PML/

RARA, or ETO/AML1 fusion, the patients were assigned to the

t(11q23), t(15;17) and t(8;21) cytogenetic groups, respectively,

even if these rearrangements were not detected by conventional

karyotyping.

Definitions of end-points and statistics. Complete remission

(CR) was defined as BM containing <5% blasts, in the

presence of regenerating normal elements and no evidence of

disease at any other site. Early death was defined as death

before or during the first 6 weeks of treatment. Patients were

considered non-responders when BM remission was not

achieved after two chemotherapy courses [after consolidation

or high dose (HD) Ara-C and mitoxantrone]. Event-free

survival (EFS) was calculated from the date of diagnosis to

the last follow-up or to the first event (i.e. failure to achieve

remission, early death, relapse, or death of any cause). Overall

survival (OS) was calculated from the date of diagnosis to the

last follow-up or to the date of death from any cause. The

last follow-up examination for all patients was performed in

April 2003.

Demographic characteristics were compared across the

groups using chi-squared test for qualitative characteristics

and t-test, Mann–Whitney test and anova were used for

quantitative characteristics. Survival curves were estimated

using the method of Kaplan and Meier (1958). Univariate

comparisons of survival curves were made by the log-rank test

(Kalbfleisch & Prentice, 1982). The procedure of Holm (1979)

was used to adjust the univariate EFS P-values for multiple

testing; unadjusted P-values are presented in the Tables. The

Cox regression model (Cox, 1972) was used for multivariate

analysis of prognostic factors for survival. P-values <0Æ05 were

considered significant.

Results

Genetic information was available for 86 of the 97 patients

(89%) with de novo AML (Table I). In eight patients, studies

were not performed, and in three, metaphases were not

obtained and FISH failed to disclose any aberration when

performed in one of these cases.

Of the 86 patients with assessable metaphases, nine had a

normal karyotype. However, molecular cytogenetics per-

formed in four of them detected an MLL split in one case,

decreasing the normal karyotype group to eight patients (9%).

In the other three patients assessed by FISH, deletion of the

ABL gene, representing possible loss of chromosome 9 or loss

of chromosome 5/5q in a subpopulation of cells, was noted in

one patient each; these were considered secondary changes.

Use of the FISH probes MLL (49 patients), ETO/AML1 (24

patients), CBFb/MYH11 (20 patients), PML/RARA (eight

patients) and BCR/ABL (37 patients) yielded new information,

not otherwise detected by conventional cytogenetics in 11

patients, resulting in a change in their cytogenetic group

classification. Specifically, MLL split was detected in nine

patients, and PML/RARA fusion in two patients. In another

four patients FISH detected del(7q) in a subpopulation of cells,

which was considered as a secondary change.

B. Stark et al

322 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337

Table I. Abnormal G-banded karyotypes and/or SKY and/or FISH findings in 78 patients with AML.

Patient number Karyotype FISH* SKY

t(11q23) (n ¼ 19)

123 51,XY,+4,+8,t(9;11)(p22;q23),+13,+16,+19/52,

idem,+del(1)(p22)

124 46,XY,t(10;11;12)(p13;q13;q13)

131 46,XY,t(1;11)(q21;q23)/46,idem,add(19)(p13)

138 46,XX,t(6;11)(q21;q23),add(18)(q?) MLL split

140 46,XX,t(X;2)(q26;q13) MLL split

149 47,XY,+8 MLL split,7q-

153 46,XY MLL split

156 46,XY,t(6;11)(q27;q23) MLL split

161 45,XX,t(1;19)(q21;p13),add(2)(q37),)10,del(11)(q23)

MLL split

167 46,XY,i(1)(q11) MLL split 46,XY,i(1)(q11),t(9;11)(p22;q23)

180 46,XX,t(11;19)(q23;p13) MLL split

181 46,XY,t(12;17)(q13;q25)/47,XY,idem,+8 MLL split 46,XY,t(12;17)(q13;q25)/47,XY,idem,+8

184 46,XX,t(9;11)(p22;q23) MLL split

185 45,XX,t(10;11)(p13;q23),)18 MLL split

188 50,XX,+8,+9,t(9;11)(p22;q23),+19,+mar/51,

idem,+i(1)(q11)/51,idem,+del(1)(p13)

MLL split

189 47,XX,+8,del(9)(q21?)/47,idem,

der(11)t(1;11)(q23;q23)/46,XX,add (7)

(p22),)13,+22.

MLL split 47,XX,+8,del(9)(q12;q22),

der(10)t(10;11)(p13;q23)/47,idem,

der(11)t(1;11)(q23;q23)/46,XX,der(7)t(3;7)

(?;p22),t(9;10)(p22;p15?),der(11)t(10;11)(p13;q23)

202 46,XX,t(11;17)(q23;q21?) MLL split

231 46,XY,add(11)(q23) MLL split 46,XY,der(11)t(9;11)

1003 51,XY,+6,+8,add(11)(q23),+14,+21,+22 MLL split 51,XY,+6,+8,t(9;11)(p22;q23),der(12)t(12;22)

(q24;q12),+14,+19,+21

t(8;21) (n ¼ 13)

104 46,XX,t(8;21)(q22;q22)

105 46,XY,t(8;21)(q22;q22)

126 46,XX,t(8;21)(q22;q22)

130 46,XY,t(8;21)(q22;q22)

134 45,X,)Y,t(8;21)(q22;q22)136 45,X,)Y,t(8;21)(q22;q22)143 46,XX,t(8;21)(q22;q22)/46,idem,add(2)(q35)/46,

idem,add(2)(q35), del(9)(q12q22),del(11)(p13)

ABLx2

147 45,X,)Y,t(8;21)(q22;q22)158 46,XY,t(8;21)(q22;q22)/46,idem,)22,+mar

208 46,X,)Y,+8,t(8;21)(q22;q22) ETO/AML1(+),)Y233 46,XX,t(8;21)(q22;q22) ETO/AML1(+)

t(15;17) (n ¼ 7)

118 47,XX,+mar

137 46,XY,inv(12)(q12;q21),t(15;17)(q22;q21)

159 46,XY,t(15;17)(q22;q21)

198 46,XX,add(3)(p?) PML/RARA(+)

215 46,XY,t(15;17)(q22;q21)

235 46,XY,t(15;17)(q22;q21),

der(12)t(8;12)(q21;p13)

8101 N.D. PML/RARA(+)

1001 46,XX,t(15;17)(q22;q21)

inv(16) (n ¼ 3)

174 46,XY,inv(16)(p13q22) CBFB split

Cytogenetics, Prognosis in Childhood Acute Myeloid Leukaemia

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337 323

Table I. Continued.

Patient number Karyotype FISH* SKY

183 48,XY,+9,inv(16)(p13q22),+22/47,

XY,del(7)(q11),del(9)(q12q22) inv (16)(p13q22),+22

CBFB split 7q-

216 46,XX,inv(16)(p13q22) CBFB split

3q21 (n ¼ 2)

127 46,XX,t(3;21)(q21;q22)/46,ide-

m,add(2)(q3?),add(4)(q23)/46,idem,add(1)(p36)

170 47,XY,inv(3)(q21q26),+21c

t(3;5) (n ¼ 2)

110 46,XY,t(3;5)(q25,q34)

152 46,XX,t(3;5)(q25,q34)

t(8p11) (n ¼ 2)

135 46,XX,t(8;19)(p11;q13)

213 46,XX,t(8;16)(p11;q13)

t(6;9) (n ¼ 1)

154 46,XY,t(6;9)(p23;q34)/46,idem,del(16)(q21)

t(1;22) (n ¼ 1)

191 46,XX,t(1;22)(p13;p13)/46,

idem,add(3)(q34),del(5)(q32)

46,XX,t(1;22)(p13;p13)/46,idem,t(3;5)(q34;q32)

7/7q- (n ¼ 5)

120 45,XX,)7141 45,XY,)7169 45,XY,)7 -CEP 7

232 45,XX,)7 -CEP 7

8102 50,XX,)2,add(5)(q35),add(7)(p11),del(7)(q21)x2,+19,+mar1,+mar2x2

7q-x2 50,XX,der(2)t(2;13)(q32;q21),der(5)t(2;5)(q31;q35),

der(6)t(6;10)(p23;?),der(7)t(7;10)(p21,?),del(7)(q21),

+del(7)(q21),+del(9)(q22),+del(9)(q22),der(13)

t(7;13)(q?;q21),+19

del(9q) (sole) (n ¼ 4)

108 46,XY,del(9)(q12q22)

155 46,XY,del(9)(q12q22) ABL x2

209 46,XY,del(9)(q12q22) ABL x2 46,XY,del(9)(q12q22)

228 46,XY,del(9)(q12q22) ABL x2

+21 (sole) (n ¼ 4)

164 47,XX,+21 21qx3

172 47,XY,+21/47,idem,del(20)(q12) 21qx3, 20q-,7q-

173 47,XY,+21/48,XY+8,+21 21qx3

1006 48,XY,+21,+21c

+8 (sole) (n ¼ 1)

171 47,XY,+8 CEP 8x3

One or two miscellaneous aberrations (n ¼ 7)

125 45,X,)X,add(14)(q?) CEP Xx1

163 47,XX,add(21)(p12),+21c

177 46,XY,del(6)(p21)/46,idem,inv(3)(q21q26) 7q-

182 46,XY,add(5)(q35?),del(6)(q21–22?) 46,XY,t(5;6)(q35;q21)

203 46,XX,del(16)(q22)

204 47,XY,t(16;17)(q22;q21),+21c/47,XY,

del(16)(q22),+21c/47,XY,+21c

217 45,X,)Y/45,X,)Y,del(21)(q21) )Y, 21q-Three and more miscellaneous aberrations (n ¼ 10)

128 45,XY,der(1)t(1;7)(p11?;q11?),der(1)t(1;13?)(p11;q11?),

der(11)t(1;11) (p22?;p15),add(3)(q26),add(7)(q11),

add(11)(q11),)13139 46,XX,del(6)(p23),del(9)(q34)/45,XX,del(6)(p23),)9,

der(22)t(9;22)(p11;p11)

ABL x1

165 56,XX,+del(2)(p22)?,+4,+6,+7,+8,+14,+19,+19,+20,

+mar

CEP 8x3

CEP 7x3

B. Stark et al

324 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337

Cytogenetic changes, incidence, and biologicalcharacteristics

The distribution of the major AML-specific cytogenetic

groups, as identified by cytogenetics and molecular cytogenetic

analysis, and their associated clinical and biological character-

istics is shown in Tables I and II.

11q23 and/or MLL gene split. These were the most frequent

changes, encountered in 19 patients (22%). In nine cases, they

were identified only by MLL splitting on FISH (Table I). The

partner chromosomes could be identified only by SKY in four

of these cases; 9p (three patients) and 10p (one patient) (Stark

et al, 2000), and in one patient, even SKY did not disclose the

translocation with 11q23. In 12 patients, translocation 11q23

was accompanied by additional structural and/or numerical

aberrations, such as extra chromosome 8 in five patients, and

extra 1q in three patients (Table I).

The clinical features of the children with t(11q23) were

unique (Table II), with extramedullary involvement in eight

patients; central nervous system (CNS) and skin involvement

in three patients each; ocular, subcutaneous masses and pleural

effusion in two patients each; and pericardial effusion and

testicular involvement in one patient each. Leucopenia

preceded the leukaemia by 6 months, and was accompanied

by severe fasciitis in one patient and extramedullary involve-

ment and leucopenia of 1 month’s duration in another. Most

of the children had an immunophenotype that was compatible

with monocytic leukaemia: positive for CD33, CD11b, CD11c,

and CD4 antigens, and occasionally CD14.

t(8;21)(q22;q22). This was the second most frequent

aberration, detected in 11 patients (13%) (Table I). It was

accompanied by loss of chromosome Y in four patients,

including one patient with an extra chromosome 8. Two

patients presented with orbital myelosarcoma, one of whom

transformed to overt leukaemia 6 weeks later. In two other

patients, refractory anaemia with excess of blasts (RAEB) or

with transformation (RAEB-t) preceded the overt leukaemia

by 1–2 months. Two additional patients showed bilineage

myelodysplastic morphological changes, with approximately

30% BM blasts at presentation. On immunophenotyping,

blasts were positive for HLA-DR in 92%, CD34 in 92%, CD13

in 75%, and CD33 in 50%. CD15 expression was documented

in five of the eight examined patients, and CD56 in four of the

seven examined patients. CD14 was negative in all patients.

t(15;17)(q22;q11–12) and/or PML/RARA rearrangement. This

aberration was observed in six patients, in four by karyotype

with FISH confirmation, and in two by FISH only (Table I). In

one of the latter, conventional cytogenetics showed add(3p);

cytogenetics was not performed in the other case. Another

(seventh) patient with an extra marker, in whom t(15;17)

could not be detected because FISH was not performed, was

nevertheless added to this group because he had FAB M3

confirmed by electron microscopy and immunophenotyping.

Immunophenotypically, all patients were negative for HLA-DR

antigen, had a low expression of CD34 antigen, and were

positive for CD13 and CD33 (Hrusak et al, 2002).

inv(16)(p13q22), CBFb/MYH11. This aberration was detected

cytogenetically and confirmed by FISH in three patients

(3Æ6%), all displaying break-apart of the CBFb gene probe

located on 16q22. FISH for inv(16) was performed in 20

additional patients [three no mitosis, three normal, three

deleted 9q, two trisomy 8, one t(6;9) and eight miscellaneous],

none of whom was positive. In one of the three positive

patients, additional numerical and structural aberrations of

del(7q), del(9q) and +22 were observed in the sideline and

confirmed by FISH (Table I). All three patients were classified

as FAB M4 with eosinophilia. Immunophenotypically, all were

strongly positive for CD34, CD13, CD33, and CD15, and only

one patient was positive for CD11b and CD4.

Monosomy 7(sole)/del(7q). This group consisted of four

patients with a simple karyotype of monosomy 7 as the sole

aberration and one patient with a complex karyotype, including

Table I. Continued.

Patient number Karyotype FISH* SKY

176 46,XY,del(2)(q31),der(12),)18,+mar 46,XY,

del(2)(q31),i(12)(q11),der(18)t(4;18)(q13;q23)

194 47,X,)X, add(11)(p13),+13,+mar/

48,XX,add(11)(p13),+13,+mar

TEL x3

CEP X x3

47,X,del(X)(p?;or q?),der(6)t(X;6) (?;q21),der(11)

t(X;11)(?;p13),+13/48,idem,+12

196 51,XX,add(2)(q35),)3,add(6)(q25),+15,+19,+22,+3mar

BCR x3

234 49,XY,+4,+6,del(7)(p11;?),+10,add(19)(q11?) 7/7q x2,

4x3, 10x3

CEP 4x3

CEP 10x3

49,XY,+4,+6,der(7)t(7;19)(p11;?),+10,der(19)

t(17;19)(q?;q11?)

1002 47,XY,+2,del(6)(q21),t(15;19)(q15;p13)

1005 46,XX,del(1)(p13)/50,XX,+del(1)(p13),+8,+8,+16 CEP 8x4 46,XX,der(1)t(1;19)(p13;p13)/50,XX,idem

+8,+8,+16

*FISH – regular font indicates the karyotype was confirmed by FISH; bold font indicates new information.

SKY, spectral karyotype ; FISH, fluorescence in situ hybridization; AML, acute myeloid leukaemia.

Cytogenetics, Prognosis in Childhood Acute Myeloid Leukaemia

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337 325

Table

II.Clinico-biologicalcharacteristicsof97

AMLchildrenincludingthe86

analysed

patientswithcytogenetic

inform

ation.

Cytogenetic

subgroup

Patients

number

(%)

Gender

(M/F)

Origin

(J/non-J)

Age

(years)

median(range)

WBC(109/l)

median(range)

Platelets(109/l)

median(range)

Extra

medl

FAB

PriorMDS

FAB

dysp

02

34

57

Totalcytogenetics

86(100)

48/38

68/18

6Æ5(0Æ0–18Æ5)

16(1–280)

62(3–408)

166

167

1825

1415

10

Norm

al8(9)

4/4

7/1

11Æ6

(6–17Æ6)

24(1–62)

80(24–298)

0–

1–

24

10

1

11q23/M

LL

19(22)

10/9

15/4

2Æ4(0–15)

53(6–280)

84(42–234)

8–

1–

215

12

0

t(8;21)

11(13)

7/4

8/3

8Æ3(2Æ9–16Æ8)

20(6–124)

40(15–120)

3–

10–

1–

–3

4

t(15;17)–

7(8)

4/3

7/0

14Æ9

(6Æ4–17Æ1)

2(2–140)

27(14–80)

0–

–7

––

–0

0

inv(16)

3(3Æ4)

2/1

3/0

12Æ2

(3Æ4–18Æ5)

92(6–115)

24(23–28)

1–

––

3–

–0

0

inv(3)/t(3;21)

2(2Æ3)

1/1

1/1

0Æ6

–(14–59)

–(15–180)

01

––

––

1§1

0

t(3;5)

2(2Æ3)

1/1

1/1

–(4Æ3–9Æ7)

–(8–10)

–(9–27)

0–

1–

––

1�1

1

t(8;16)

2(2Æ3)

0/2

2/0

–(15–15Æ4)

–(3–4)

–(22–26)

0–

––

2–

–0

0

t(6;9)

1(1Æ1)

1/0

1/0

13Æ0

1864

0–

1–

––

–0

0

t(1;22)

1(1Æ1)

0/1

1/0

0Æ3

2568

0–

––

––

10

0

)7/7q-(sole,m

ain)

5(5Æ8)

2/3

5/0

9Æ0(0Æ5–14Æ7)

22(1–30)

23(4–54)

02*

––

11

11

1

9q-(sole)

4(4Æ6)

4/0

4/0

9Æ9(2Æ5–16Æ1)

48(14–84)

120(46–255)

0–

2–

2–

–1

0

+21

(sole)

4(4Æ6)

3/1

2/2

2Æ3(1Æ6–4Æ5)

10(6–18)

14(3–38)

0–

––

–1

3§3

0

+8(sole)

1(1Æ1)

1/0

1/0

12Æ0

284

0–

––

––

10

0

Miscellaneous<3abnorm

alities

7(8)

4/3

4/3

2Æ8(1Æ5–16Æ9)

10(2–180)

61(14–99)

12�

––

21

2�§

22

Miscellaneous‡3

abnorm

alities

9(10)

4/5

6/3

5Æ5(0Æ2–18Æ4)

13(3–73)

104(27–408)

31�

––

23

21

1

Nomitoses

32/1

1/2

7Æ4(1Æ5–15Æ5)

13(8–82)

67(30–444)

1–

––

2–

10

0

Notdone

83/5

3/5

5Æ0(0Æ4–20)

23(5–320)

43(6–380)

12�

2–

21

10

0

TotalAML

9753/44

72/25

6Æ9(0–20)

18(1–320)

61(3–408)

188

187

2226

1615

10

*FABM1,

onepatient.

�FABM6,

onepatient.

�Basophylic

leukaem

iabyelectronmicroscope,fourpatientspreviouslyreported

(Shvidelet

al,2003).

§Downsyndrome,fourpatients(twoofthem

inthemiscellaneousgroup).

–OnepatientwithFABM3andmarkerchromosomewas

added

tothis

group.J/non-J,Jewish/non-Jew

ish;Extra

medl,extram

edullaryinvolvem

ent;FABdysp,FABdysplasia;

AML,acute

myeloid

leukaem

ia;FAB,French-A

merican-British;MDS,

myelodysplastic

syndrome.

B. Stark et al

326 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337

del(7q) · 2, confirmed by FISH and SKY (Table I). In another

five patients, loss of 7q was considered a secondary change: one

patient (no. 128) with incomplete complex miscellaneous

karyotype with add(7)(q11) in whom FISH failed; one (no. 183)

with the primary aberration inv(16) (15 metaphases) and

del(7q11) identified cytogenetically in six of the 15 metaphases

and by FISH in a subpopulation (32% of cells); and three

patients with the loss of submicroscopic 7q31 detected only by

FISH in a subpopulation (20–45%) of interphase nuclei (50–

100), including one patient with MLL split (no. 149), one with

del(6p) (no. 177), and one with trisomy 21 (no. 172). FISH for

7/7q was normal in the other 37 patients [including 13 with

miscellaneous aberrations, six with 11q23 abnormalities, and

three with del(9q), normal, or no mitosis]. FAB subtypes varied

and refractory anaemia preceded the leukaemic transformation

by 3 years in one patient (Table II).

del(9)(q12;q22). Interstitial deletion of 9q12q22 as the sole

karyotypic abnormality was found in four patients. FISH

analysis was performed in three patients, and the BCR/ABL

probes disclosed the remainder of the two ABL genes located on

9q34, in accordance with the interstitial deletion. FISH with all

AML panel probes showed a normal pattern. Another five

patients had a visible deletion of (9q) in addition to other

primary structural aberrations (Table I): in a subclone of t(8;21),

inv(16), t(10;11); in a complex karyotype with del(7q); and in a

subclone of del(6)(p23). The deletion in the first four patients

seemed to be interstitial, with preservation of the two ABL genes

(by FISH), and was more distal with a loss of ABL gene locus in

the last patient. There was no difference inmedian age and white

blood cell count (WBC) between the solitary del(9q) group and

the whole series. However, three patients in the solitary del(9q)

group had a higher platelet count (over 100 · 109/l). Except for

some lymph node enlargement, no extramedullary involvement

was noted. FAB M2 and M4 leukaemias were found in two

patients each, with strong myeloperoxidase staining, and Auer

rods in all. One patient presented with RAEB, transforming

within 3 weeks to leukaemia with 30% BM blasts. All four

patients were positive for HLA-DR, CD34, and CD33, and three

of four, for CD13 and CD7.

Trisomy 21. Trisomy 21 as the sole acquired aberration was

noted in four patients, three of them with M7 leukaemia, one of

these with Down syndrome, and the other two were identical

twins, described in an earlier report (Stark et al, 2002). FISH

analysis, performed in three patients, confirmed the cytogenetic

findings and ruled out an underlying cryptic MLL split, del(7q)

and del(5q). Extra chromosome 21 as a secondary change was

observed in one patient in a sideline of monosomy 7.

Trisomy 8. One patient had trisomy 8 as the sole abnormality,

confirmed by FISH using the whole AML probe panel.

Secondary extra chromosome 8 was seen in another nine

patients; six had the primary 11q23 aberrations, one t(8;21),

one trisomy 21, one miscellaneous (Table I).

Rare recurrent chromosomal abnormalities. The

t(8;16)(p11;p13) and its variant t(8;19)(p11;q13) (Stark et al,

1995) were detected as the sole aberration in one patient each

with FAB M4-5 morphology with erythrophagocytosis.

Abnormal (3q) was detected in four patients.

t(3;5)(q25;q34) was the sole aberration detected in two

patients; one had erythroleukaemia (M6) and the other had

RAEB, which transformed after a few weeks to leukaemia;

t(3;5)(q25;q34) also appeared in a sideline to the primary

t(1;22) (Table I). In the other two patients, both infants,

t(3;21)(q21;q22) and inv(3)(q21q26) were detected in one

each. The latter infant, with Down syndrome, had M7

leukaemia following a short transient myeloproliferative dis-

order. In addition, inv(3)(q21;q26) was detected in a subclone

of del(6p).

Other rare recurrent abnormalities were t(6;9)(p23;q34) in

one patient with FAB M2 leukaemia, and t(1;22)(p13;q13) in

one infant with M7.

Miscellaneous. Miscellaneous clonal abnormalities were initially

found by cytogenetics in 26 patients. With the application of

FISH, MLL split was detected in seven of the 22 patients tested,

and PML/RARA rearrangements in one of the four patients

tested. One additional patient, with an extra marker

chromosome in whom FISH failed, was transferred to the

t(15;17) group because he had proven FAB M3 morphology.

This left only 16 patients with unclassified miscellaneous

aberrations (Table I): seven patients with one or two structural

or numerical aberrations and nine patients with three or more

structural changes. FISH analysis showed a 7q deletion in one of

the 14 patients examined, no 5 or 5q deletion in 13 patients and

no ABL/BCR rearrangement in 12 patients examined. The

clinical and biological characteristics of the miscellaneous group

are detailed in Table II. Two young children had Down

syndrome and M7 leukaemia. There was a relatively high

proportion of M0 (three), and M7 (five) subtypes. In three

patients (two with M0, one M7) basophilic leukaemia was

detected by electron microscopy, two of them (nos 203, 204)

have been previously reported (Shvidel et al, 2003).

There were four children with Down syndrome (Table I, nos

170, 1006, 163, 204) with chromosome abnormalities:

inv(3)(q21;q26), acquired +21, add(21)(p12), t(16;17)(q22;

q21) respectively. Their ages ranged between 0Æ6 and 1Æ6 years,

all with M7 leukaemia.

Outcome by cytogenetic subgroups and clinicobiologicalparameters

Overall, outcome analysis of the 94 patients with newly

diagnosed AML treated with BFM-based protocols yielded, at a

median follow-up of 69 months (range 4–177), a remission

rate of 93%, with an estimated probability of 4-year EFS of

55Æ7 ± 5% and OS of 59Æ9 ± 5% (Table III). Among the 83

patients who were evaluated cytogenetically, EFS was

52Æ1 ± 6% and OS 57Æ1 ± 6%.

Cytogenetics, Prognosis in Childhood Acute Myeloid Leukaemia

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337 327

Treatment protocols, type of BMT, and outcome by

cytogenetic subgroup are demonstrated in Table III and

Fig 1. The 4-year EFS in patients with a normal karyotype

and in patients with t(8;21), most of whom were treated with

the earlier protocol, was 62% and 60% respectively. Of the

seven patients with t(15;17), one died early in induction from

disseminated intravascular coagulopathy and one relapsed

4 years after diagnosis, leading to a drop in EFS from 85Æ7% to

57Æ1 ± 25%; however, since the latter patient was salvaged, OS

at 4 years was 85Æ7 ± 13%. All three patients with inv(16) are

alive in first remission. Of the 19 patients with 11q23

abnormalities, all but one who lived longer than 6 months

underwent allogeneic transplantation (four patients) or autol-

ogous transplantation (nine patients), according to HLA-

matched sibling availability. The 4-year EFS in this subgroup

was 33Æ8 ± 11% and OS 44Æ6 ± 11%. Similar results were

noted for the subgroup of six patients with t(9;11) (EFS

22 ± 19%) and the other t(11q;23) (EFS 38 ± 13%)

(P ¼ 0Æ24). Of the five patients with monosomy 7, two did

not achieve remission, and two relapsed within 1 year. Only

one, transplanted early, is alive in remission after 9 months. All

three patients with solitary del(9q) relapsed within 18 months

and died. Patients with miscellaneous abnormalities had an

EFS of 60Æ2 ± 12%. No difference was found between those

with more or less than three abnormalities (EFS 66Æ6 ± 15%

vs. 57Æ1 ± 18% respectively, P ¼ 0Æ9). The appearance of

del(9q), del(7q), and trisomy 8 as additional secondary

changes did not add any prognostic significance to the primary

aberrations.

A complex karyotype of three or more unrelated cytogenetic

abnormalities, excluding the t(8;21), inv(16), t(15;17), and

normal karyotype groups, had a trend-level effect on 4-year

EFS: 51 ± 8% for the 37 patients with a simple karyotype vs.

29 ± 12% for the 17 patients with a complex one (P ¼ 0Æ08).Within the complex karyotype group, the 1-year EFS for the 12

patients with three to four abnormalities was 33 ± 15%, and

for the five patients with five or more abnormalities, it was

20 ± 18% (P ¼ 0Æ43). A complex karyotype with three or

more abnormalities had a poor prognostic impact within the

t(11q23) group, with a 4-year EFS of 0 vs. 50Æ3 ± 14% for

those with simple t(11q23) (P ¼ 0Æ019).This retrospective series covered two main treatment

periods: the early one, in which treatment was based on the

AML-BFM-87 protocol (Creutzig et al, 1999), and the later

one, when treatment was based on the BFM-93, -98

protocols (Creutzig et al, 2001). The protocols differed

mainly in the postinduction timing of HD Ara-C: in the

BFM-87, it followed the 6 weeks of consolidation, whereas

in the BFM-93/-98, it was administered earlier, immediately

after induction. In addition, in the second period, autolo-

gous and allogeneic BMT were performed in a higher

percentage of patients: 34% and 27% vs. 25% and 6% in the

Table III. Treatment and outcome of 97 acute myeloid leukaemia (AML) children by cytogenetic subgroups.

Cytogenetic subgroup

Number

of patients

Treatment Outcome

BFM

BMT

(auto/allo)87 93/98 Other* No CR Relp

4-year EFS�(% ± SE)

4-year OS�(% ± SE)

Total cytogenetics 86 44 39 3 23/13 5 31 52Æ1 ± 6 57Æ1 ± 6

Normal 8 7 1 0 3/0 0 3 62Æ5 ± 17 62Æ5 ± 17

11q23/MLL 19 9 10 0 9/4 1 11 33Æ8 ± 11 44Æ6 ± 11

t(8;21) 11 9 2 0 1/2 0 4§ 60Æ0 ± 15 60Æ0 ± 15

t(15;17) 7 2 5 0 0/0 1� 1 57Æ1 ± 25 85Æ7 ± 13

inv(16) 3 0 3 0 2/0 0 0 100 100

t(8;16) 2 1 1 0 2/0 0 0

)7/7q-(sole,main) 5 3 2 0 0/1 3 2 0 0

9q- (sole) 4 2 1 1 0/0 0 3 0 0

+21 (sole) 4 1 2 1 1/2 0 0 100 100

Miscellaneous <3 abnormalities 7 2 5 0 2/0 0 3 57Æ1 ± 18 57Æ1 ± 18

Miscellaneous ‡3 abnormalities 9 3 6 0 1/4 2 1 66Æ6 ± 15 36Æ4 ± 27

No mitoses 3 2 1 0 1/0 0 1 66Æ6 ± 27 66Æ6 ± 27

Not done 8 6 2 0 3/1 0 1 87Æ5 ± 11 87Æ5 ± 11

Total AML 97 52 42 3 27/14 5 33 55Æ7 ± 5 59Æ9 ± 5

*Another protocol was used for three patients, who were treated elsewhere and lost to follow-up. They were not included in the assessment of EFS and

OS.

�EFS (event-free survival) and OS (overall survival) were calculated for the 94 newly diagnosed patients treated with the modified AML-BFM-87, -

93/-98 protocols.

�Early death within 9 d.

§One patient died in first remission from chronic GVHD.

B. Stark et al

328 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337

first period respectively (P ¼ 0Æ005). There was no signifi-

cant difference in the distribution of the patient character-

istics (gender, origin, WBC, platelet count and cytogenetic

risk groups) between the two periods, except for a

marginal difference in FAB subtypes with some higher

incidence of M5, M7 of 33% and 19%, respectively in the

second period, compared with 23% and 11% in the first

period (P ¼ 0Æ08).With respect to outcome, remission was achieved in 95% of

the patients treated by the later protocols compared with 92%

of those treated by the early one (P ¼ 0Æ69); death in remission

was 0% vs. 2Æ3% (P ¼ 0Æ4), cumulative incidence of relapse

was 31Æ6 ± 8% vs. 44Æ7 ± 7% (P ¼ 0Æ35). The 4-year EFS was

65Æ1 ± 8% in the second period, with a median follow-up of

49 months (range 4–74), and 49Æ8 ± 7% in the first period,

with a median follow-up of 125 months (range 26–177), with

no significant difference (P ¼ 0Æ24) at the time of analysis, and

the same for OS, 66Æ6 ± 8% vs. 55Æ8 ± 7% respectively

(P ¼ 0Æ35).Because of the relatively small number of children and the

non-significant differences in their presenting features and

outcome, the results of both treatment periods were combined

for univariate analysis of prognosis (Table IV). Male gender,

FAB dysplasia, WBC 50 · 109/l to 100 · 109/l, and platelet

count over 50 · 109/l were associated with lower EFS. FAB M5

leukaemia was associated with a trend of worse 4-year EFS

compared with the rest of the group (42% vs. 61%, P ¼ 0Æ12).The small subgroup of FAB M7 had a better, but statistically

insignificant, 4-year EFS (79% vs. 51%, P ¼ 0Æ12). Three (nos170, 163, 204) of the four patients with Down syndrome were

treated with modified BFM protocols, one of them underwent

an autotransplant. In one patient (no. 204) the disease

recurred and the child died.

Multivariate analysis was performed with the variables

found to be significant on univariate analysis: cytogenetic risk

group (unfavourable versus others), gender, platelet count

(>50 · 109/l), FAB dysplasia, and WBC (<50, 50–100, >100).

Of these, unfavourable cytogenetic risk group [P < 0Æ001,

relative risk (RR) 4Æ08, 95% confidence interval (CI): 1Æ92–8Æ68]; FAB dysplasia (P ¼ 0Æ007, RR 4Æ00, 95% CI: 1Æ62–9Æ88);and male gender (P ¼ 0Æ029, RR 2Æ12, 95% CI: 1Æ06–4Æ26),remained significant adverse prognostic factors.

Outcome by cytogenic risk groups

On the basis of outcome in terms of EFS probability, and the

previously published cytogenetic risk classification in adults

Fig 1. Overall survival by cytogenetic abnormalities.

Table IV. Prognostic factors of the 94 newly diagnosed children

treated with the modified acute myeloid leukaemia Berlin-Frankfurt-

Munster (AML-BFM) protocols.

Variable

Patients/events

(n)

4-year EFS

(% ± SE)

P-value

(log-rank)

Total patients 94/39 55Æ7 ± 5 –

Gender

Female 44/12 70Æ8 ± 7 0Æ006Male 50/27 42Æ3 ± 7

Origin

Non-Jew 24/7 70Æ8 ± 9 0Æ15Jew 70/32 50Æ0 ± 6

Age (years)

<2 24/10 58Æ0 ± 10 0Æ58<10 36/17 49Æ8 ± 8

<15 22/9 55Æ1 ± 11

>15 12/3 67Æ3 ± 16

WBC (·109/l)<50 71/27 59Æ5 ± 6 0Æ02<100 12/8 27Æ8 ± 13

>100 11/4 60Æ6 ± 15

Platelets (·109/l)<50 44/12 72Æ3 ± 7 0Æ011‡50 48/26 41Æ2 ± 7

FAB type

0, 1 8/3 58Æ3 ± 18 0Æ482 18/8 55Æ6 ± 12

3 7/2 57Æ1 ± 25

4 21/9 53Æ2 ± 11

5 26/14 42Æ0 ± 11

6, 7 14/3 78Æ6 ± 11

FAB-dysplasia

Negative 84/32 59Æ4 ± 6 0Æ010Positive 10/7 22Æ5 ± 14

Prior MDS

Negative 80/29 61Æ3 ± 6 0Æ007Positive 14/10 23Æ8 ± 12

Cytog. risk groups* (83 patients)

Favourable 21/6 66Æ8 ± 11 0Æ005Intermediate 21/6 71Æ1 ± 10

Unfavourable 41/25 33Æ6 ± 8

*Cytogenetic risk groups: favourable: t(8;21), inv(16), t(15;17), FAB

M3.

Intermediate: normal, t(8;16), t(1;22), +21(sole), miscellaneous <3

abnormalities.

Unfavourable: t(11q23), del(9q)(sole), abn(3q), t(6;9), )7/7q), mis-

cellaneous ‡3 abnormalities.

Cytogenetics, Prognosis in Childhood Acute Myeloid Leukaemia

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337 329

(Grimwade et al, 1998; Slovak et al, 2000; Byrd et al, 2002),

patients in the present series (albeit small in number – 83 were

cytogenetically assessable and treated with the modified BFM

protocols) could be assigned to three risk categories: favour-

able – 21 patients (25%) with t(8;21), t(15;17), or inv(16) with

and without secondary abnormalities; intermediate – 21 pati-

ents (25%) with normal karyotype, +21(sole), t(8;16), t(1;22),

or miscellaneous simple (<3 abnormalities); and unfavourable

– 41 patients (50%) with t(11q23), t(6;9), abn(3q), del(9q)

(sole), )7/7q-, miscellaneous complex (‡3 abnormalities). The

4-year EFS of the three cytogenetic risk groups was

66Æ8 ± 11%, 71Æ1 ± 10%, and 33Æ6 ± 8%, respectively

(P ¼ 0Æ005, significance at the 5% level after applying Holm’s

procedure), and the OS was 74Æ4 ± 10%, 71Æ1 ± 10%, and

37Æ4 ± 8% respectively (P ¼ 0Æ02) (Fig 2). All together, in the

favourable and intermediate cytogenetic risk groups, consisting

half of the patients, the 4-year EFS and OS was 69Æ6 ± 7% and

72Æ8 ± 7%, respectively, compared with the other half (unfa-

vourable group), with 33Æ6% and 40Æ8 ± 8%, respectively

(P ¼ 0Æ0012, P ¼ 0Æ0005 respectively).

Discussion

The incidence of cytogenetic de novo AML subgroups and their

clinico-biological characteristics and outcome were retrospec-

tively analysed in children that were diagnosed at a referral

paediatric centre in Israel.

On the basis of our use of the FISH technique, 11% of the

patients were reassigned, leading to a more reliable subgroup

classification. The major change was the decrease in the

proportion of patients in the miscellaneous group, from 30 to

18%, and a nearly twofold increase in the 11q23/MLL group,

from 11 to 22%. Other changes included a decrease in the

normal karyotype (10–9%) and solitary +8 groups (2–1%),

and an increase in the t(15;17)/PML/RARA (4Æ5–7%). No

cryptic inv(16) or Philadelphia chromosome was detected by

FISH in the miscellaneous and normal groups, or among the

patients with changes considered to be secondary. Comparable

findings, with up to 41% of the MLL translocations detected

only by FISH, were reported in an acute leukaemia series from

Korea (Kim et al, 2002). In another recent large adult AML

study with a high yield of assessable metaphases, 33% of

t(11q23) cases, 23% of t(8;21) cases, 12% of inv(16) cases, and

5% of t(15;17) cases were identified by FISH only (Frohling

et al, 2002).

Incidence

The incidence rates of most of the cytogenetic groups in our

study in general were similar to the larger childhood series

from Europe and the USA (Table V).

A normal karyotype was found by conventional cytogenetics

in 10% of patients, decreasing to 9% following the use of the

FISH panel in half the patients. Despite the possible effect of

methodological difference on the incidence determination of

normal karyotype, no occult chromosome anomalies were

detected by a comprehensive FISH panel in 55 de novo AML

adult patients with a normal karyotype, suggesting a ‘true’

normal karyotype group (Cuneo et al, 2002). This group

includes among others, patients with non-random molecular

changes, such as Flt3 internal tandem duplication (Levis &

Small, 2003; Zwaan et al, 2003). The normal cytogenetic group

in all reported studies was consistently smaller in children with

AML (9–29%) as in our study, than in adults (38–51%)

(Table V), and increased with age (Grimwade et al, 1998,

2001).

The most frequent translocations affected chromosome

band 11q23, detected in 22% of the cohort. This high rate

was attributed to the addition of the FISH method in our

series. A similar rate of 18–19% was reported in the large US

and Nordic series (Martinez-Climent et al, 1995b; Raimondi

et al, 1999; Forestier et al, 2003) and 14% and 15% by the

Children’s Cancer Group (CCG) 213 and Medical Research

Council (MRC) 10/12 series respectively (Wells et al, 2002;

Harrison et al, 2003) (Table V). The incidence of 11q23

abnormalities varies with age, being high in infants (40–60%)

(Pui et al, 2000; Chessells et al, 2002) and younger patients,

and decreasing to 2Æ8–7% in adults (Table VI) (Schoch et al,

2003). The most common partner chromosome to 11q23 is

chromosome 9, as in our series, with t(9;11) reported in 7–9%

of the paediatric series (Table V) (Martinez-Climent et al,

1995b; Raimondi et al, 1999; Forestier et al, 2003; Harrison

et al, 2003). In our series and in others, monocytic M5

leukaemia with extramedullary involvement was highly corre-

lated with t(11q23). In two of our patients, granulocytic

sarcoma preceded the overt leukaemia by a few weeks, as

previously described by others (Johansson et al, 2000).

The t(8;21) was the second most common abnormality,

detected in 13%, close to the rates reported in childhood series

from the USA and UK (Raimondi et al, 1999; Wells et al, 2002;

Harrison et al, 2003) (Table V), and higher than in the Nordic

series (Forestier et al, 2003). Rates in adults are 6–8%, and

Fig 2. Overall survival by cytogenetic risk groups.

B. Stark et al

330 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337

Table

V.Frequency

ofcytogenetic

abnorm

alitiescompared

withrecentlargechildhoodandadult

de

nov

oAMLseries.

Children

Adults

Present

study

No.(%

)

Harrison

etal

(2003)

MRC10/12

No.(%

)

Forestier

etal

(2003)

NOPHO

93

No.(%

)

Wells

etal

(2002)

CCG

213

No.(%

)

Raimondi

etal

(1999)

POG

8821

No.(%

)

Martinez-C

liment

etal

(1995a)

No.(%

)

Grimwade

etal

(1998,

2001)

MRC10/11

No.(%

)

Mrozek

etal

(2001),

Byrd

etal

(2002)

CALGB8461

No.(%

)

Slovak

etal

(2000)

SWOG/ECOG

No.(%

)

Totalnumber

ofpatients

97255

498

610

120

1795

808

Age

(years)

<20

<15

<18

<21

<21

<20

15–55

15–86

16–55

Totalanalysed

86(88)

808

239(93)

269(54)

478(78)

115(96)

1244

1213

(77)

609(80)

Norm

al8(9)

179(22)

67(28)

77(29)

109(23)

17(15)

560(45)

582(48)

t(8;21)

11(13)

94(12)

18(7)

35(13)

56(12)

9(8)

–(7)

81(7)

50(8)

t(15;17)

7(8)

67(8)

8(3)

NI

55(11)

12(10)

–(13)

88(7)

27(4)

inv(16)/t(16;16)

3(3)

46(6)

13(5)

17(5)

28(6)

9(8)

–(4)

96(8)

53(9)

t(11q23)*

19(22)

119(15)

44(18)

38(14)

88(18)

21(19)

–(3)

54(5)

42(7)

t(9;11)

6(7)

51(6)

21(9)

35(7)

10(9)

–(<1)

27(3)

t(8;16)

2(2)

–(1)

3(1)

–(<1)

)7/del(7q)

5(6)

59(7)

8(3)

11(4)

9(2)

6(5)

–(6)

95(8)

del(9q)(sole)

4(4)

14(4)

1–(1)

11(1)

17(3)

+21(sole)

4(4)

1(0)

7(1)

5(<1)

Miscellaneous

16(18)

89(19)

NI,notincluded;AML,acute

myeloid

leukaem

ia.

*t(11q23)withallpartner

chromosomes

includingchromosome9.

Cytogenetics, Prognosis in Childhood Acute Myeloid Leukaemia

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337 331

drop to 1–2% at older ages (Slovak et al, 2000; Grimwade et al,

2001; Schoch et al, 2001a). In adults, racial differences were

noted, with an increased incidence of t(8;21) in African-

Americans compared with Caucasians (Sekeres et al, 2002) and

in Asian-Japanese compared with Australians (Nakase et al,

2000). The t(8;21) associated with FAB M2 morphology, in

rare cases, may occur with MDS (Kojima et al, 1998; Felice

et al, 2000), or MDS may precede the leukaemia (Taj et al,

1995) or leukaemia may be associated with dysplastic changes

(Haferlach et al, 1996; Nakamura et al, 1997). This was noted

in a few of our patients as well. Granulocytic sarcoma in the

orbit, the epidural and malar regions, as documented in two of

our patients, were described in higher proportions in children

from Argentina (Felice et al, 2000) and black children from

South Africa (Schwyzer et al, 1998).

The frequency of t(15;17) in our series was in the range of

the childhood series from the USA and UK, namely, 8–11%

(Table V). A lower (3%) frequency was reported in the Nordic

series (Forestier et al, 2003), and a higher one (17%) in the

paediatric Italian series (Biondi et al, 1994). Geographic

variations and a higher prevalence was also observed in adult

Latino and African-American series (Douer et al, 1996; Sekeres

et al, 2002). The absence of non-Jewish representation in this

subgroup is of note (Table II) and may suggest an ethnic

variation.

The chromosome 16 aberration inv(16)(p13;q22) occurred

in only 3% of our cohort. Testing by FISH in another 19

patients with normal or miscellaneous aberrations was negat-

ive. In the USA, UK and Nordic childhood series, rates ranged

from 5 to 8% (Table V). This difference warrants a future

study of a larger cohort for possible geographic heterogeneity,

because of the ethnic genetic make-up of specific gene

polymorphisms. For example, the C609T polymorphism in

the NQO1 (quinone oxyreductase 1) gene, which lowers the

activity of the enzyme, thereby leading to a decrease in quinone

detoxification, was found to be associated with an increased

risk of inv(16) in adult AML (Smith et al, 2001).

The recurrent balanced translocations t(8;16), t(6;9), t(3;5)

and t(1;22) occurred rarely, together accounting for 6% of the

cases, in line with the findings of Raimondi et al (1999). As in

other childhood series, we did not detect the rare Philadelphia

chromosome, even by FISH (performed on 41 patients).

The incidence of the numerical changes of monosomy 7 and

7q- and extra chromosome 21 as sole aberrations was also

within the range of previous childhood studies (Table V).

Trisomy 8 as the sole aberration was detected in only one

patient. A similar rate was noted in the series of Raimondi et al

(1999), lower than in other paediatric series (Forestier et al,

2003) and adult series (Byrd et al, 1998; Wolman et al, 2002).

In another of our patients, an apparent solitary +8 was later

determined to be a MLL split, by FISH.

In the present series, solitary int del(9q) was observed in 4%

of patients. This group was mentioned briefly in only one

paediatric series (Harrison et al, 2003), and the reported rate

in adult series is <1% (Grimwade et al, 1998, 2002; Byrd et al,

2002). Like others (Ferrara et al, 1996; Wan et al, 2003), we

noted an association of del(9q) with FAB M2 and M4, or

preceding MDS (RAEB-t), presence of Auer rods and CD7 and

CD34 expression.

The rare miscellaneous abnormalities accounted for 18% of

our cases, as reported also by Raimondi et al (1999) and the

Nordic group (Forestier et al, 2003). This group included

some aberrations described anecdotally by Raimondi et al

(1999), such as del(16)(q23) and t(16;17)(q24;q11), in addi-

tion to t(X;6) (Dastugue et al, 1997), t(1;19) (Tchinda et al,

2002), or -Y(sole), which accounted for 1Æ5% of the adult series

of the Cancer and Leukemia Group B (CALGB) (Byrd et al,

2002).

Treatment, prognosis and cytogenetic risk groups

The children in the present series were treated with the AML-

BFM-87, -93/-98 protocols, with extended TIT injections

replacing cranial irradiation. Overall, CR was achieved in 95%;

the 4-year EFS and OS were 55Æ7 ± 5%, and 59Æ9 ± 5%

respectively. These results are in line with the improved EFS

reported by multicentre paediatric clinical studies (Arceci,

2002): BFM-93, 51% at 5 years (Creutzig et al, 2001); MRC 10

and 12, 48% and 52% at 5 years (Webb et al, 2001; Gibson

et al, 2002); Nordic Society of Pediatric Oncology and

Hematology (NOPHO) 93, 49% at 7 years (Lie et al, 2003);

and CCG 2891 intensive timing, 49% survival at 8 years

(Woods et al, 2001).

The cytogenetic subgroups had different impacts on prog-

nosis. Patients with t(8;21), treated with the modified BFM-87

protocol had a relatively favourable prognosis, with a 4-year

EFS and OS of 60% each, close to the rates achieved in other

series based on the AML-BFM-87 (Felice et al, 2000), the

NOPHO 93 (EFS 56%) and the Pediatric Oncology Group

(POG) 8821 (EFS 45%) (Raimondi et al, 1999). An OS of 81%

was recently reported in the MRC 10/12 (Harrison et al, 2003).

All these protocols included HD Ara-C. The benefit of

repetitive HD Ara-C in adult t(8;21) AML was previously

proven by the CALGB group (with an OS of 76% compared

with 44% in patients receiving lower doses) (Bloomfield et al,

1998; Byrd et al, 1999, 2002), and confirmed by the UK series

(OS 69%) (Grimwade et al, 1998). Some studies claimed that

within the t(8;21) group, additional secondary del(9q) or loss

of sex chromosome may have an adverse impact on outcome

(Haferlach et al, 1996; Schoch et al, 1996; Slovak et al, 2000),

although this has not been supported by others (Grimwade

et al, 1998; Raimondi et al, 1999; Byrd et al, 2002; Harrison

et al, 2003) or our series. In the present series, relapse occurred

in three of four patients with myelodysplastic changes, whereas

dysplastic features had no prognostic significance in the adult

t(8;21) series of Haferlach et al (1996).

Patients with the other core-binding factor type leukaemia

with inv(16), are considered a favourable prognostic group. In

our series too, this group had a very good outcome, and all

three patients are alive in first CR. Small paediatric series with

B. Stark et al

332 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337

inv(16) AML and a larger adult series demonstrated an EFS of

58–76% (Raimondi et al, 1999; Razzouk et al, 2001; Wells

et al, 2002; Lie et al, 2003) and an OS of 57–81% respectively

(Grimwade et al, 1998; Byrd et al, 2002; Delaunay et al, 2003;

Harrison et al, 2003). All protocols but one (Delaunay et al,

2003) used HD Ara-C.

Our third favourable group was t(15;17), treated with all-

trans-retinoic acid (ATRA) combined with BFM-based che-

motherapy; EFS was 57% and OS was 83%. Similar results

were achieved with the NOPHO 93 (Lie et al, 2003) and the

MRC 10/12 (Harrison et al, 2003), and adult protocols based

on concurrent ATRA and chemotherapy in induction and

maintenance (Tallman et al, 2002).

The normal karyotype group is considered to be of

intermediate risk by all studies, with an EFS of 37–43% in

paediatric series (Raimondi et al, 1999; Wells et al, 2002;

Harrison et al, 2003; Lie et al, 2003) and an OS of 24–45% in

adult series (Grimwade et al, 1998, 2002; Byrd et al, 2002). In

our normal group, EFS and OS were both 62%.

The outcome of the largest group of t(11q23)/MLL split was

poor, with a 4-year EFS of 34% and OS of 45%. There was no

difference between the patients with t(9;11) and the rest of the

t(11q23) group. While the CCG 213 (Wells et al, 2002), and

POG 8821 series (Raimondi et al, 1999) noted similar results, a

few other paediatric centres showed a better outcome for the

t(9;11) group, with an EFS of 56–85% (Martinez-Climent et al,

1995b; Rubnitz et al, 2002; Lie et al, 2003). In the adult

CALGB 8461 series, t(9;11) also carried a better prognosis [OS

41% intermediate risk vs. 0% high risk for the rest of t(11q23)

group] (Mrozek et al, 2001; Byrd et al, 2002). In the adult

MRC 10/12 trial, OS was 42% for the whole abn(11q23) group,

with no significant difference from t(9;11) (5-year OS, 56%)

and the other 11q23 partners (Grimwade et al, 2002; Harrison

et al, 2003). These authors classified the whole group of

abn(11q23) as intermediate risk. However, the Southwest

Oncology Group (SWOG) study classified abn(11q23) to be an

unfavourable risk (Slovak et al, 2000). In one in vitro study

using the cell kill methyl-thiazoltetrazolium (MTT) assay,

t(9;11) blasts showed higher sensitivity to Ara-C, etoposide,

anthracyclines and 2-chlorodeoxyadenosine than the other

11q23 translocations and the other AMLs (Zwaan et al, 2002).

In the present study, within the t(11q23) group, the presence

of a complex karyotype with three or more secondary

aberrations conferred a fatal outcome, as opposed to 50%

survival for those with fewer or no additional chromosomal

changes. However, in the CALGB 8461 study, neither complex

karyotype nor secondary aberrations affected the outcome of

patients with t(9;11) (Byrd et al, 2002).

The small group with monosomy 7 fared extremely poorly.

Only one of four patients is currently in remission following

early BMT, which is in line with the unfavourable classification

in other paediatric and adult series (Grimwade et al, 1998,

2002; Byrd et al, 2002; Wells et al, 2002).

The small del(9q) (sole) group was categorized in the adult

MRC 10/12 series and in the CALGB trial as intermediate risk,

with an OS of 36–55% (Grimwade et al, 1998, 2002; Byrd et al,

2002), and by the SWOG study (Slovak et al, 2000) as

unfavourable risk. In one recent paediatric series, survival was

not different from the rest of the group (Harrison et al, 2003).

In the present series, all three patients relapsed within

5–20 months; none underwent transplantation.

Trisomy 21 was considered an intermediate risk group in the

adult CALGB and MRC series. However, a high remission rate

was achieved in the paediatric POG series (Raimondi et al,

1999). In the present series, all three children with trisomy 21

are in long-term remission following the BFM protocol and

BMT (Stark et al, 2002).

Abn(3q), especially inv(3)(q21;q26) or t(3;3), is categorized

in all adult series as an extremely poor prognostic group

(Grimwade et al, 1998, 2002; Byrd et al, 2002). Patients with

t(3;5)(q25;q34) in the POG series fared poorly (Raimondi

et al, 1999), but in a recent adult series, OS was 45%

(Grimwade et al, 2002). In the present study, two of four

children with abn(3q) are in remission.

The child with t(6;9) in this series relapsed. Accordingly, all

earlier paediatric and adult series classified t(6;9) as a worst

prognostic group (Raimondi et al, 1999; Byrd et al, 2002;

Grimwade et al, 2002).

The recurrent t(8;16) remains unclassified in the large series.

However, in the few affected patients in the POG series

(Raimondi et al, 1999), and on review of other reports on a

limited number of patients (Stark et al, 1995), outcome

seemed poor. By contrast, in the present series, both patients

were cured.

The infant with t(1;22) in our sample was cured with the

BFM-98 protocol and autologous BMT. In an earlier review

(Bernstein et al, 2000), and in the POG series (Raimondi et al,

1999), the t(1;22) group had an adverse outcome, whereas in a

recent French report of childhood megakaryocytic leukaemia,

t(1;22) carried a 3-year EFS of 50% (Dastugue et al, 2002).

The del(16)(q22) was detected in one patient who relapsed.

In some classifications, del(16)(q22) was combined with the

favourable inv(16), whereas in others, it was excluded from the

inv(16) group because it often differed in both characteristics

and outcome, with an OS of only 10% (Marlton et al, 1995;

Byrd et al, 2002).

Complex karyotype, in all but the favourable risk group, was

considered in all studies as a predictor of adverse outcome.

Complex karyotype was defined by the MRC as ‡5 unrelated

abnormalities (Grimwade et al, 1998), and by the CALGB,

SWOG and German groups as ‡3 abnormalities (Slovak et al,

2000; Schoch et al, 2001b; Byrd et al, 2002). In our series, EFS

was 52% for <3 abnormalities, 33% for ‡3 abnormalities, and

20% for ‡5 abnormalities.

On the basis of outcome in the present series and the

published criteria (Bloomfield et al, 1997; Grimwade et al,

1998; Slovak et al, 2000; Byrd et al, 2002), we identified three

cytogenetic risk groups. The well-defined favourable risk group

consisted of t(8;21), t(15,17), and inv(16), even when accom-

panied by secondary changes or complex karyotype (Byrd et al,

Cytogenetics, Prognosis in Childhood Acute Myeloid Leukaemia

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 320–337 333

2002), excluding del(16q). This group accounted for about

25% of our sample. Four-year EFS and OS were 67% and 74%,

respectively, comparable with the OS of 65% in the adult MRC

10 series (Grimwade et al, 1998) and the 55% in the CALGB

study (Byrd et al, 2002).

The definition of the intermediate risk group is more

variable. All series included a normal karyotype. The CALGB

and MRC studies also included trisomy 21(sole) and

del(9q)(sole). Like in the SWOG study, we moved the

del(9q) from the intermediate to the unfavourable risk group,

and added t(8;16), t(1;22) (previously unclassified) and

miscellaneous with <3 abnormal aberrations. The MRC also

included the miscellaneous abnormalities in the intermediate

group. Our intermediate risk group accounted for 25% of the

cohort. Outcome was similar to the favourable risk group,

with both the 4-year EFS and OS being 71%. In a recent

paediatric MRC 12 series, survival from the time of CR was

79% (Gibson et al, 2002). In adults, the intermediate risk

group is much larger (46–61%) because of the larger normal-

karyotype subgroup and the inclusion of either t(9;11) or all

abn(11q23) by the CALGB and MRC series, respectively,

which report a 5-year OS of 24–41% (Grimwade et al, 1998;

Byrd et al, 2002).

Our unfavourable risk group comprised t(11q23), as in the

SWOG study (Slovak et al, 2000), in addition to t(6;9),

abn(3q), )7/7q-, del(9q)(sole) and miscellaneous complex

karyotype (‡3 unrelated abnormalities) – similar, for the most

part, to the adult series (Slovak et al, 2000; Grimwade et al,

2001; Byrd et al, 2002). It accounted for 50% of the sample,

and EFS and OS were 33% and 37% respectively. The poor

prognosis was confirmed also by multivariate analysis. In

adults, the unfavourable risk group consists of 10–30% of

patients, with a very poor OS of 5–14% (Slovak et al, 2000;

Grimwade et al, 2001; Byrd et al, 2002).

All together, two main cytogenetic risk categories of similar

size could be expected; one consisting of patients in the

favourable and intermediate groups, in whom EFS was 70%,

compared with the remaining patients with an unfavourable

prognosis and EFS of 33% (P ¼ 0Æ0012).In conclusion, the cytogenetic analysis of childhood AML

using FISH yielded a higher number of patients with

cytogenetic information and a more accurate risk classification,

with optimal clinical relevance. Although modern therapeutic

protocols have led to a better outcome in AML, cytogenetic

aberrations still show a significant impact on prognosis, as

demonstrated also in our relatively small series. Within the

favourable/intermediate risk groups, additional cytogenetic or

molecular genetic markers may improve prediction of relapse

and treatment stratification. In the higher risk group, earlier

BMT and new modalities directed at overcoming drug

resistance are needed.

The recently acquired knowledge on gene expression profiles

in relation to cytogenetic subsets will further refine risk

assessment and optimize targeted therapy.

Acknowledgments

The authors wish to thank Dina Kugel for her expert data

management; Meira Zoldan for the morphological and cyto-

chemistry staining; and Yona Kodman for the immunophen-

otypic assessment. Authors also thank Ilana Gelernter and Hila

Shemer of the Statistical Laboratory of Tel Aviv University for

their excellent assistance with the statistical analysis (I.G.) and

database management (H.S.), and Gloria Ginzach and Marian

Propp for their editorial and secretarial assistance.

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