Karyotype complexity and prognosis in acute myeloid leukemia

Abstract

A complex aberrant karyotype consisting of multiple unrelated cytogenetic abnormalities is associated with poor prognosis in patients with acute myeloid leukemia (AML). The European Leukemia Net classification and the UK Medical Research Council recommendation provide prognostic categories that differ in the definition of unbalanced aberrations as well as the number of single aberrations. The aim of this study on 3526 AML patients was to redefine and validate a cutoff for karyotype complexity in AML with regard to adverse prognosis. Our study demonstrated that (1) patients with a pure hyperdiploid karyotype have an adverse risk irrespective of the number of chromosomal gains, (2) patients with translocation t(9;11)(p21∼22;q23) have an intermediate risk independent of the number of additional aberrations, (3) patients with [= or >, slanted]4 abnormalities have an adverse risk per se and (4) patients with three aberrations in the absence of abnormalities of strong influence (hyperdiploid karyotype, t(9;11)(p21∼22;q23), CBF-AML, unique adverse-risk aberrations) have borderline intermediate/adverse risk with a reduced overall survival compared with patients with a normal karyotype.

OPEN

Citation: Blood Cancer Journal (2016) 6, e386; doi:http://dx.doi.org/10.1038/bcj.2015.114

Web End =10.1038/bcj.2015.114

http://www.nature.com/bcj

Web End =www.nature.com/bcj

F Stlzel1,15, B Mohr1,15, M Kramer1, U Oelschlgel1, T Bochtler2, WE Berdel3, M Kaufmann4, CD Baldus5, K Schfer-Eckart6,R Stuhlmann7, H Einsele8, SW Krause9, H Serve10, M Hnel11, R Herbst11, A Neubauer12, K Sohlbach12, J Mayer13, JM Middeke1, U Platzbecker1, M Schaich14, A Krmer2, C Rllig1, J Schetelig1, M Bornhuser1 and G Ehninger1

A complex aberrant karyotype consisting of multiple unrelated cytogenetic abnormalities is associated with poor prognosis in patients with acute myeloid leukemia (AML). The European Leukemia Net classication and the UK Medical Research Council recommendation provide prognostic categories that differ in the denition of unbalanced aberrations as well as the numberof single aberrations. The aim of this study on 3526 AML patients was to redene and validate a cutoff for karyotype complexity in AML with regard to adverse prognosis. Our study demonstrated that (1) patients with a pure hyperdiploid karyotype havean adverse risk irrespective of the number of chromosomal gains, (2) patients with translocation t(9;11)(p2122;q23) have an

intermediate risk independent of the number of additional aberrations, (3) patients with 4 abnormalities have an adverse risk per se and (4) patients with three aberrations in the absence of abnormalities of strong inuence (hyperdiploid karyotype, t(9;11)

(p2122;q23), CBF-AML, unique adverse-risk aberrations) have borderline intermediate/adverse risk with a reduced overall survival

Blood Cancer Journal (2016) 6, e386; doi:http://dx.doi.org/10.1038/bcj.2015.114

Web End =10.1038/bcj.2015.114 ; published online 15 January 2016

INTRODUCTION

The term complex aberrant is designated to describe karyotypes with multiple unrelated cytogenetic abnormalities. In acute myeloid leukemia (AML), 1014% of all AML patients, and up to 23% among older AML patients, have karyotypes with 3 aberrations.14 These karyotypes with 3 aberrations are classied as adverse genetic risk according to the recommendations of the

European Leukemia Net (ELN).1 However, the UK National Cancer

Research Institute Adult Leukaemia Working Group (abbreviated as MRC for Medical Research Council) classication requires 4 abnormalities as an informative cutoff for adverse prognosis.5

Beyond the 3 vs 4 cutoff discordance, the impact of the so-called pure hyperdiploid karyotype (HDK) without structural aberrations or monosomies has not been addressed conclusively.6,7 Further complicating, the denition of unique adverse-risk abnormalities, which dene adverse risk per se, is not fully congruent in both classication systems, with some abnormalities conferring adverse risk according to the ELN but not the MRC and vice versa. Given this heterogeneity, further exploration of complexity seems desirable for several reasons. First, it has been demonstrated that in the adverse-risk group, some patients with certain chromosomal abnormalities fare even worse than others when receiving standard treatment regimens for adverse-risk patients.812 Second, better individual risk prognostication and uniformly dened

adverse-risk group allocation are required in order to homogeneously compare treatment regimens at different institutions.

The aim of this study was to dene the optimized cutoff of complexity in adult AML in the context of the number of unrelated aberrations (3 vs 4) as well as to dene the impact of the pure

HDK within these groups. Therefore, we evaluated the survival of 417 intensively treated adult non-APL and non CBF-AML patients with complex aberrant karyotypes out of 3526 AML patients who were included in three prospective, randomized, multicenter treatment trials of the Study Alliance Leukemia.

PATIENTS AND METHODS Patient population

The databases of three prospective, randomized trials of the Study Alliance Leukemia, which enrolled a total of 3526 non-APL, intensively treated AML patients between February 1996 and November 2009, were reviewed for patients with multiple cytogenetic aberrations (3) as well as normal karyotype (NK as a control group). The studies were approved by the institutional review boards of all participating centers of the Study Alliance Leukemia in agreement with the Declaration of Helsinki and registered with the National Clinical Trial numbers 00180115 (AML96 trial), 00180102 (AML2003 trial) and 00180167 (AML60+ trial). Written informed consent had been obtained from each patient.

At diagnosis, chromosome analyses were performed on bone marrow and/or peripheral blood samples using standard techniques, including

1Medizinische Klinik und Poliklinik I, Universittsklinikum Carl Gustav Carus der TU Dresden, Dresden, Germany; 2Abteilung Innere Medizin V, Universitt Heidelberg, Heidelberg, Germany; 3Medizinische Klinik A, Universittsklinikum Mnster, Mnster, Germany; 4Robert Bosch Hospital, Stuttgart, Germany; 5CharitUniversittsmedizin Berlin, Berlin, Germany; 6Asklepios Klinik St Georg, Hamburg, Germany; 7Asklepios Klinik St Georg, Hamburg, Germany; 8Medizinische Klinik und Poliklinik II, Universittsklinikum Wrzburg, Wrzburg, Germany; 9Medizinische Klinik 5, Universittsklinikum Erlangen, Erlangen, Germany; 10Medizinische Klinik II, Klinikum der J.W. Goethe Universitt, Frankfurt, Germany;

11Klinik fr Innere Medizin III, Klinikum Chemnitz, Chemnitz, Germany; 12Kliniken fr Innere Medizin, Hmatologie/Onkologie und Immunologie, Universittsklinikum Marburg, Marburg, Germany; 13University Hospital Brno, Brno, Czech Republic and 14Klinik fr Hmatologie und Onkologie, Rems-Murr-Kliniken, Winnenden, Germany. Correspondence: Dr F Stlzel, Department of Internal Medicine I, University Hospital Carl Gustav Carus, Technical University Dresden, Fetscherstr. 74, Dresden 01307, Germany.

E-mail: mailto:[email protected]

Web End [email protected]

15These authors contributed equally to this work.

This study was presented in part as oral presentation at the 55th ASH Annual Meeting of the American Society of Hematology, 710 December 2013, New Orleans, LA, USA. Received 4 December 2015; accepted 14 December 2015

ORIGINAL ARTICLE

Karyotype complexity and prognosis in acute myeloid leukemia

compared with patients with a normal karyotype.

Karyotype complexity in AML F Stlzel et al

2

short-term cultures as reported recently.13 Karyotype description was performed in accordance with the International System for Human Cytogenetic Nomenclature criteria.14 According to the denition of the MRC, a balanced translocation, for example, t(8;21)(q22;q22), was dened as a single abnormality, because the two breaks and fusions lead to one active chimeric fusion protein. A balanced translocation involving more than two chromosomes was also regarded as a single abnormality. Trisomies or monosomies were regarded as single abnormalities, whereas the gain of two chromosomes, even if they were identical (e.g., tetrasomy 8), was regarded as two abnormalities. Unbalanced translocations leading to gain and loss of chromosomal material were counted as two abnormalities.5 For instance, a derivative chromosome der(7)t(1;7)(q21;q22) is characterized by a partial monosomy 7q as well as a partial trisomy 1q. In this manner, an isochromosome i(17)(q10) results in two aberrations, that is, monosomy 17p and trisomy 17q. The monosomal karyotype (MK) was dened by the presence of two or more distinct autosomal chromosome monosomies or a single autosomal chromosome monosomy in the presence of one or more structural chromosomal abnormalities.8

Cytogenetic denitionsOut of the 3526 patients, a total of 2007 patients with either a complex karyotype or a normal karyotype were identied for further analyses (n = 1590 patients with NK; n = 417 patients with 3 aberrations which accounted for 30% of the patients in the AML96 trial and 29% of the patients in the AML2003/60+ trialsreferring to those patients for whom an aberrant karyotype was diagnosed). Patient characteristics are summarized in Tables 1A and B. The median follow-up time for all patients was 6.2 years (interquartile range, 4.58 years). Core-binding factor AML patients (CBF-AML, t(8;21)(q22;q22), inv(16)(p13q22), t(16;16) (p13:q22)) were excluded since additional chromosomal abnormalities even if they resulted in complex aberrant karyotypes have no or little impact on the outcome of patients with favorable-risk CBF-AML5 and could be conrmed with our CBF-AML patients.11,15 However, previous results demonstrated an independent inuence of the pure HDK on patients outcome worsening overall survival (OS) and event-free survival signicantly.15 The scoring criterion for pure HDK performed in our analyses was dened by (i) gains of whole chromosomes (e.g., trisomies, tetrasomies), (ii) no additional structural aberrations and (iii) no monosomies.

The following distinct cytogenetic features were included as possible candidates of strong inuence: (I) three or four unrelated aberrations,

(II) specic adverse-risk aberrations that induce an adverse outcome per se: unique adverse-risk aberrations dened by the ELN and the MRC were applied in this study which were, in detail, inv(3)(q21q26), t(3;3)(q21;q26), abnl(3q) except t(3;5)(q2125;q31q35), 5, del(5q), add(5q), 7, del

(7q), add(7q), t(6;9)(p23;q34), t(v;11)(v;q23) except t(9;11)(p2122;q23),

17 and abnl(17p),1,5 (III) AML with recurrent genetic abnormalities according to the World Health Organization. This category includes the recurrent abnormalities t(9;11)(p22;q23), t(6;9)(p23;q34), inv(3)(q21q26.2)/ t(3;3)(q21;q26.2), and t(1;22)(p13;q13).16 Translocation t(6;9) and inv(3)/ t(3;3) are specic adverse-risk aberrations and therefore already included in that category. Translocation t(1;22) is a rare aberration with n = 1 patient. Therefore, no further investigation was possible. Thus, only t(9;11) remained as a feature of particular interest, and (IV) pure HDK with gains of whole chromosomes (e.g., trisomies, tetrasomies), but without additional structural aberrations or monosomies.

The distinct cytogenetic features were considered with the following groups of complex aberrant patients: (a) HDK, (b) t(9;11), (c) complex karyotypes with three unrelated aberrations without specic adverse-risk aberrations, without HDK, without t(9;11) (CK3), (d) complex karyotypes with three unrelated aberrations with at least one specic adverse-risk aberration, without HDK, without t(9;11) (CK3+adv), (e) complex karyotypes with 4 unrelated aberrations without specic adverse-risk aberrations, without HDK, without t(9;11) (CK4) and (f) complex karyotypes with 4 unrelated aberrations with at least one specic adverse-risk aberration, without HDK, without t(9;11) (CK4+adv). Comprehensive owcharts of the distinct groups of complex aberrant karyotypes are depicted in Figures 1a and b.

In order to investigate the inuence of an MK in the complex aberrant situation, patients were divided into (a) patients with three unrelated aberrations without MK (CK3 MK), (b) patients with three unrelated aberrations with MK (CK3+MK), (c) patients with 4 unrelated aberrations without MK (CK4 MK) and (d) patients with 4 unrelated aberrations with

MK (CK4+MK).

Treatment protocolsDetailed treatment descriptions of the three trials were reported previously.13,17,18 In brief, the AML96 trial enrolled adult patients without

age restriction, whereas the AML2003 trial included patients up to 60 years of age, and the AML60+ trial patients above the age of 60 years. Apart from double induction chemotherapy administered to patients aged 60 years, all three protocols involved a risk-adapted consolidation strategy, including HLA-compatible related or unrelated allogeneic hematopoietic

Table 1A. Patient characteristics

NK HDK t(9;11) CK3 CK4 CK+adv n = 1590 n = 20 n = 10 n = 19 n = 35 n = 333

Median age (range) 56 (1787) 60 (3776) 40 (2960) 56 (3378) 56 (1880) 61 (1582) Gender, male (%) 741 (47) 10 (50) 4 (40) 11 (58) 17 (49) 172 (52)

De novo AML (%) 1318 (83) 16 (80) 9 (90) 12 (63) 26 (74) 221 (66) Median WBC (Gpt/l), range 16 (0.2453) 12 (0.9192) 24 (1212) 10 (0.873) 7.5 (0.6468) 4.1 (0.2197) Median LDH (U/l), range 427 (1016 560) 513 (1003058) 881 (2762997) 261 (275489) 660 (937938) 362 (974160)

Table 1B. Patient characteristics

CK3+MK CK3 MK CK4+MK CK4 MK n = 22 n = 43 n = 228 n = 124

Median age (range) 63 (3275) 62 (2482) 61 (1582) 58 (1881) Gender, male (%) 12 (54) 24 (56) 118 (52) 60 (48)

De novo AML (%) 14 (64) 30 (70) 149 (65) 91 (73) Median WBC (Gpt/l), range 7 (0.898) 11 (0.6156) 4 (0.2468) 5 (0.4212) Median LDH (U/l), range 406 (1352540) 371 (275489) 361 (977938) 414 (933946)

Abbreviations: NK, normal karyotype (control group); HDK, hyperdiploid karyotype; CK3, complex aberrant patients with three unrelated abnormalities without HDK, without t(9;11), and without adverse-risk abnormalities; CK4, complex aberrant patients with four or more unrelated abnormalities without HDK, without t(9;11), and without adverse-risk abnormalities; CK+adv, complex aberrant patients with three or more aberrations of which at least one aberration was at specic adverse risk per se with exclusion of patients with t(9;11) or HDK; CK3+MK, patients with MK and with three unrelated aberrations; CK3 MK, patients with three unrelated aberrations without MK; CK4+MK, patients with four or more unrelated aberrations with MK; CK4 MK, patients with four or more unrelated aberrations without MK; WBC, white blood count; LDH, lactate dehydrogenase.

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Complex aberrant with three unrelated aberrations, n = 65 patients

minus

n = 9 HDK patients

n= 56 patients

minus

n= 35 patients with adverse risk abnormalities (CK3+adv)

n= 2 patients with t(9;11)

CK3, n = 19 patients

Complex aberrant with four or more unrelated aberrations, n = 352 patients

minus

n = 11 HDK patients

n = 341 patients

minus

n = 298 patients with adverse risk abnormalities (CK4+adv)

n = 8 patients with t(9;11)

CK4, n=35 patients

Figure 1. The owcharts depicts all patients characterized by 3 (a) and 4 unrelated abnormalities (b), respectively, and end with the groups CK3 and CK4. Patients with (i) pure hyperdiploid karyotype (HDK), (ii) adverse-risk aberrations and (iii) t(9;11) were removed constituting distinct groups because of their strong specic inuence on outcome per se. All of the complex aberrant patients with adverse abnormalities per se are summarized to the CK +adv group (n = 333) resulting from n = 35 CK3+adv and n = 298 CK4+adv patients. CK3+adv consists of complex aberrant karyotypes with three unrelated aberrations with at least one specic adverse-risk aberration, without HDK, without t(9;11) and CK4+adv consists of complex karyotypes with 4 unrelated aberrations with at least one specic adverse-risk aberration, without HDK, without t(9;11).

stem cell transplantation for intermediate-risk patients with a sibling donor and adverse-risk patients with a matched donor. In the AML2003 trial, patients were randomized up-front to undergo allogeneic hematopoietic stem cell transplantation early after induction chemotherapy-induced aplasia or during rst remission in dened adverse-risk situations.19

Statistical analysisComplete remission was dened according to the standard consensus criteria.20 OS was measured from the date of entering the study to the date of event (death) or last follow-up and was reported for the whole cohort. The KaplanMeier method was used to estimate the probability for OS. Median OS were provided for all end points with 95% condence intervals (CIs). The stratied log rank test was used for univariate comparison of OS. The stratication variable was the study generation.

To determine the prognostic inuence of the distinct cytogenetic groups independent of age, WBC, serum lactate dehydrogenase levels at baseline, and type of AML (de novo AML, AML with preceding myelodysplastic syndrome, therapy-related AML) as covariates, a stratied multivariable Cox regression analysis for OS was performed. Stratication variable again was study generation. Because of its informative character, allogeneic hematopoietic stem cell transplantation was not censored. All statistical analyses were performed using SPSS version 19.0.1 (SPSS Inc, Chicago, IL, USA) and the R environment for statistical computing version 2.15.3.21

RESULTSThe following cytogenetic subgroups were analyzed for their inuence on OS.

Hyperdiploid karyotypeOut of 417 patients with 3 aberrations, 20 patients displayed a pure HDK with a range of 4980 chromosomes (median, 50 chromosomes) without other abnormalities. Nine patients with HDK had 3 trisomies and 11 patients had 4 trisomies. The most frequent chromosomes involved in the formation of HDK were chromosomes 8, 4, 13, 9, 10, 21 and 22 (in decreasing frequency), present in at least more than 20% of all patients with HDK. Additionally, tetrasomies 4, 8, 13, 14, 20 and 21, each detected in 12 patients, as well as pentasomies 13, 21 and 22, each detected in one patient, were found.

The median OS for these patients was 4.6 months (95% CI, 017.4) (Figure 2a). The multivariable Cox regression including age, WBC, lactate dehydrogenase and type of AML showed that HDK was an independent prognostic factor for OS (HR 2.2; 95% CI,1.43.5; P = 0.001). There was no inuence of the number of trisomies or tetrasomies on survival. Patients with three trisomies and patients with four or more trisomies/tetrasomies had a similar probabilities of OS (P = NS, data not shown). Furthermore, we compared HDK patients with patients with cytogenetic adverse-risk criteria according to the ELN/MRC classications (Figure 2b). OS did not differ signicantly (HR 0.6; 95% CI, 0.41.1; P = 0.082). For the adverse control group (CK+adv, n = 333) no further distinction between CK3+adv (n = 35) and CK4+adv (n = 298) was performed since HDK patients with 3 or 4 aberrations had similar survival.

Additional data resulting from univariate comparisons (log rank test) regarding OS and data resulting from multivariable Cox regression analysis of these patients and the patients documented below are summarized in the supplement (Supplementary Tables S1 and S2).

t(9;11) and other WHO recurrent cytogenetic abnormalitiesThe following WHO recurrent cytogenetic aberrations were detected within the complex aberrant karyotypes of the patients analyzed: t(9;11)(p2122;q23) (n = 10 patients); t(6;9)(p23;q34)

(n = 3 patients); inv(3)(q21q26.2)/t(3;3)(q21;q26.2) (n = 9 patients) and t(1;22)(p13;q13) (n = 1 patient). Patients with t(9;11) had an OS similar to patients with NK with a median survival of 23 months (95% CI, 1333.1) (Figure 2c) Interestingly, 8 out of the 10 patients had a karyotype grouped into the CK4 cohort and 2 patients had additional specic adverse-risk abnormalities.

Complex karyotype with three independent aberrations (CK3) vs complex karyotype with four or more independent aberrations (CK4)

To delineate the best cutoff of complexity, we analyzed survival data from patients with 3 or 4 independent aberrations (CK3, n = 19 and CK4, n = 35, respectively) who furthermore did not have any of the adverse-risk cytogenetic aberrations, without HDK, and without a t(9;11) (Figure 2d). CK4 patients without t(9;11) and without adverse-risk criteria had a signicant inferior OS as compared with the control group. Multivariable Cox regression analysis conrmed these observations for OS showing that CK4 (without adverse-risk abnormalities, without t(9;11) and without HDK) is an independent adverse prognostic factor for OS in comparison with NK (HR, 2.2; 95% CI, 1.53.3; Po0.001).

Interestingly, OS of CK3 patients without t(9;11) and the adverse-risk criteria was reduced only slightly (HR, 1.6; 95% CI, 0.92.7; P = 0.078) while there was no signicant effect for event-free survival at all (data not shown).

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Karyotype complexity in AML

F Stlzel et al

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MK/unique adverse-risk aberrationsMany patients shared the cytogenetic features MK and specic adverse-risk aberrations (Tables 2A and B and Figure 3). In the group of patients with three aberrations, 34% patients (n = 22) had an MK, whereas in the group of patients with 4 aberrations, 65%

patients (n = 228) had an MK, resulting in a total of 250 patients with MK. A total of 333 patients had specic adverse-risk aberrations (54%, n = 35, in those patients with 3 aberrations and 82%, n = 298 in those patients with 4 aberrations).

The frequency of chromosome 17p abnormalities in these groups was 11% and 38%, respectively. Of the patients belonging to the CK3+MK group (n = 22), 64% had additional specic adverse-risk aberrations (n = 14), whereas 95% (n = 217) of the patients belonging to the CK4+MK group (n = 228) had additional specic adverse-risk aberrations. Because MK is often caused by monosomy of chromosomes 5, 7 or 17, we investigated the frequencies of these

monosomies in the MK group. A total of 45% of patients with CK3+MK as well as 66% patients with CK4+MK harbored at least one of the above monosomies. When investigating the inuence of MK, all four

Table 2A. Karyotype details in patients with 3 or 4 aberrations without specic consideration of other abnormalities (see also

Supplementary Figures 1A and B)

3 aberrations n = 65

4 aberrations n = 352

Hyperdiploid karyotype (%) 9 (14) 11 (3) Adverse-risk abnormality (%) 35 (54) 298 (82) abnl(17p) (%) 7 (11) 135 (38) Monosomal karyotype (%) 22 (34) 228 (65)

Abbreviation: abnl(17p), abnormality of chromosome 17p.

Overall Survival

Overall Survival

100

80

60

40

20

HDK, n=20

CK+adv, n=333

NK, n=1590

HDK, n=20

Probability (%)

Probability (%)

0

0 0 1 2 3 4 5

100

80

60

40

20

0

1 2 3 4 5

Time (years)

Time (years)

C

95% CI) 5.8

med HR* p va

dian OS, months * (95% CI)alue*

NK.2 (18.3 24)

--

HDK 4.6 (0 17.4) 2.2 (1.4 3.5) 0.001

(95% CI) 21.

media HR* ( p valu

an OS, months (9 (95% CI)ue*

CK+adv (4.9 6.7) 4 --

HDK 4.6 (0 17.4) 0.6 (0.4 1.1) 0.082

Overall Survival

Overall Survival

100

80

60

40

20

NK, n=1590 NK, n=1590

t(9;11), n=10 CK3, n=19

CK4, n=35

Probability (%)

Probability (%)

0

0

0 0 1 2 3 4 5

Time (years)

100

80

60

40

20

1 2 3 4 5

Time (years)

med HR* p va

dian OS, months * (95% CI)alue*

(95% CI) 21.

NK.2 (18.3 24)

--

t(9;11)23 (13 33.1) 1.5 (0.7 3.1) 0.328

media HR* ( p valu

an OS, months (9 (95% CI)ue*

CK3.8 (2.8 16.7) 1.6 (0.9 2.7)0.078

CK4 6.1 (3.8 8.4) 2.2 (1.5 3.3) < 0.001

95% CI) 21.2

NK

(18.3 24) 9--

Figure 2. (a) Overall survival (OS) of patients with normal karyotype (NK) and with pure hyperdiploid karyotype (HDK) AML from the time of diagnosis. (b) OS of patients with HDK and with complex aberrant karyotype AML with 3 aberrations (CK+adv) of which at least one aberration predicts an adverse risk per se, but without HDK and t(9;11) from the time of diagnosis. (c) OS of patients with NK and with complex aberrant karyotype with t(9;11)(p2122;q23) from the time of diagnosis. (d) OS of patients with NK, with complex aberrant karyotype with three unrelated abnormalities but without HDK, t(9;11), and specic adverse-risk aberrations (CK3), with complex aberrant karyotype with 4 unrelated abnormalities but without HDK, t(9;11), and specic adverse-risk aberrations (CK4). Median OS is depicted in the respective table.

Cox regression (*, hazard ratio (HR), P-value) was performed applying age, WBC, LDH and the type of AML (AML with antecedent myelodysplastic syndrome and therapy-related AML) as co-variables. Abbreviation: LDH, lactate dehydrogenase.

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subgroups that is, CK3 MK, CK3+MK, CK4 MK and CK4+MK, fared worse with regard to OS than the NK control group (Figures 4a and b).

The additional inuence of specic adverse-risk aberrations on outcome was more pronounced in patients with CK4 as compared with patients with CK3 (CK3 vs CK3+adv and CK4 vs CK4+adv; Supplementary Figures S1a and b). Median OS in months were 9.8 (95% CI, 2.816.7) for CK3, 11 months (95% CI, 616.1) for CK3+adv, 6.1 months (95% CI, 3.88.4) for CK4 and 5.4 months

(95% CI, 4.56.2) for CK4+adv, respectively. The hazard ratios for the risk of death in these groups compared with NK patients in the multivariable analysis were 1.6 for CK3 (95% CI, 0.92.7; P = 0.085),1.6 for CK3+adv (95% CI, 1.12.3; P = 0.010), 2.3 for CK4 (95% CI,1.63.3; Po0.001), and 3.3 for CK4+adv (95% CI, 2.83.8; Po0.001).

DISCUSSIONOur analysis of the prognostic role of karyotype complexity (3 vs 4) in adult AML patients demonstrates that a higher number of patients have 4 (84%) aberrations than 3 (16%)

aberrations. Patients with 4 aberrations clearly fare worse than NK patients, which could be demonstrated for the distinct groups

CK4, CK4+adv, CK4 MK and CK4+MK. Of additional unfavorable inuence on OS was the existence of unique adverse-risk aberrations (risk of death in comparison with NK patients for CK4 HR, 2.3; 95% CI, 1.63.3 vs CK4+adv HR, 3.3; 95% CI 2.83.8) or an MK (CK4 MK HR, 2.7; 95% CI, 2.23.3 vs CK4+MK HR, 3.3; 95% CI, 2.83.9). Patients with three unrelated aberrations had a worse outcome than NK patients, too. However, the effect was less impressive than in patients with 4 aberrations.

The MRC data demonstrated that the level of karyotype complexity has little impact on the outcome in patients already having at least one of the independent abnormalities conferring favorable or adverse risk. Additionally, the MRC reported that in patients lacking any of these independent adverse-risk abnormalities, the presence of 4 unrelated changes was found to provide the most informative cutoff, predicting a signicantly inferior prognosis.5

The ELN classication scheme allocated patients with the recurring aberration t(9;11)(p22;q23) to the intermediate-II genetic risk group.1 Our data conrm this stratication showing that the t(9;11)(p2122;q23) confers an intermediate risk even with an accompanying complex karyotype.

Our study implies that AML patients with an HDK, specically those without additional monosomies or structural aberrations, should be allocated to an adverse-risk category because of the signicant inuence on survival in comparison with NK patients

Table 2B. Karyotype details in patients with 3 or 4 aberrations and with a monosomal karyotype

CK3+MK CK4+MK n = 22 n = 228

Monosomal karyotype without adverse-risk abnormality (%)

8 (36) 11 (5)

Monosomal karyotype with 5, 7 or 17 (%) 10 (45) 151 (66)

Abbreviations: CK3+MK, patients with a monosomal karyotype and a complex karyotype with three unrelated aberrations; CK4+MK, patients with a monosomal karyotype and a complex karyotype with 4 unrelated aberrations.

3 aberrations n = 56

4 aberrations n = 341

Figure 3. Distribution of karyotype abnormalities in patients with three abnormalities exclusive of an HDK and patients with 4 abnormalities exclusive of an HDK. The karyotype abnormalities in these patients include monosomal karyotype (MK), unique adverse-risk risk karyotype only, unique adverse-risk risk karyotype in combination with MK, and patients without unique adverse-risk risk karyotype and MK. Patients with pure HDK are excluded.

Overall Survival Overall Survival

NK, n=1590 NK, n=1590 CK3-MK, n=43CK3+MK, n=22 CK4+MK, n=228

CK4-MK, n=124

Probability (%)

100

80

60

40

20

Probability (%)

100

80

60

40

20

0

0 0 1 2 3 4 5 0 1 2 3 4 5

Time (years) Time (years)

N

K

med

(95% HR* p va

dian OS, months

% CI)* (95% CI) alue*

NK 21.2 (18.3 24)

--

CK3-MK 10.5 (4.9 16.1

1.7 (1.2 2.3)0.003

CK3+MK) 10.1 (6.1 14.

1.7 (1.1 2.80.020

median OS, m

1) (95% CI) HR* (95% CI) p value*

months 21.2 (18

) --

8.3 24) 6.2 (4

- 2.7 (2 - <

CK4-MK CK4+MK .5 7.8) 5.5 (

.2 3.3) 3.3 (0.001 <

4.4 6.7)

2.8 3.9) < 0.001

Figure 4. Overall survival (OS) from the time of diagnosis of patients with normal karyotype (NK), (a) with complex aberrant karyotype with three unrelated abnormalities but without MK (CK3 MK), with complex aberrant karyotype with three unrelated abnormalities and MK (CK3 +MK), (b) with complex aberrant karyotype with 4 unrelated abnormalities but without MK (CK4 MK), and with complex aberrant karyotype with 4 unrelated abnormalities and MK (CK4+MK). Median OS are depicted in the respective tables. Cox regression (*, hazard ratio (HR), P-value) was performed applying age, WBC, LDH and the type of AML (AML with antecedent myelodysplastic syndrome and therapy-related AML) as co-variables. Abbreviation: LDH, lactate dehydrogenase.

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(OS HR, 2.2; 95% CI, 1.43.5; P = 0.001). Recently, the impact of hyperdiploidy in AML patients was published independently, emphasizing the impact of this category.6 Although the authors identied a similar, obviously non-random pattern of chromosomal gains comparable to our data, they applied a different approach by also including patients with monosomies in their HDK group with numerical changes, whereas we addressed the impact of pure hyperdiploidy separately without including patients with structural abnormalities and patients with loss of chromosomes. An analysis of the French Groupe Francophone de Cytogenetique Hematologique investigated 38 AML patients with high HDKs restricted to karyotypes with only high hyperdiploidy with 49 or more chromosomes.7 Because of the inclusion of children in the analysis by Luquet et al., a different methodology and different statistical methods being applied, a direct comparison with our results is not possible.

To date, it is not clear whether additional monosomies or structural aberrations occur earlier in the development of AML or whether one or the other abnormality acts as the driver or the passenger aberration. Although gains of additional chromosomes appear to represent the result of clonal evolution caused by the failure of the mitotic machinery rather than an initiating event in AML, we suggest classifying patients with pure HDK as a distinct category, excluding monosomies and structural abnormalities.

The introduction of the MK category by Breems et al. offered the application of a further criterion in risk stratication of AML patients showing that MK identies a subset of patients with very poor prognosis, which has been conrmed by other groups.5,8,22

Our study, which was restricted to the distinct population of complex aberrant patients, showed a relevant inuence on prognosis for the presence of an MK only in the CK4 situation with an increased risk of death for patients with CK4+MK as compared with patients with CK4 MK. In patients with CK3 MK or with CK3+MK the risk of death was superimposable. Additionally, our data conrm that distinct cytogenetic features that accompany other abnormalities have a strong inuence on outcome and must be considered independently, for example, patients with t(9;11) conferring intermediate risk.

A consistent denition of adverse-risk complex aberrant karyotype AML appears to be warranted. Here, we conrm a karyotype with 4 aberrations and a pure HDK as impressive adverse-risk abnormalities in AML. Patients with three unrelated aberrations fare worse than NK, too, but with an OS classifying between the 4 patients and the intermediate NK. This is an important nding that may help to stratify patients to individual optimized treatment strategies and may therefore lead to improved individual survival prognostication. Therefore, based on our ndings, we suggest the following re-classication of cytogenetic risk: (1) favorable risk: CBF-AML; (2) intermediate risk: normal karyotype, t(9;11); (3) adverse risk: three aberrations without specic adverse-risk abnormalities, without HDK; (4) very adverse risk: 4 aberrations, HDK, specic adverse-risk abnormalities, as dened by the ELN and MRC.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGEMENTS

The contributions of all patients, nurses, lab technicians and physicians in the trials of the SAL study group are highly appreciated.

AUTHOR CONTRIBUTIONSGE and MB provided nancial and administrative support. FS and BM designed the study. FS, BM and MK collected the cytogenetic and clinical data. FS, BM, MK, UO, TB, WEB, MK, CDB, KS-E, RS, HE, SK, HS, MH, RH, AN, KS, JMM, UP, MS,

AK, CR, JS, MB and GE analyzed and interpreted the data. All authors contributed to the writing process and approved the nal manuscript.

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