Ann Hematol DOI 10.1007/s00277-015-2494-9

ORIGINAL ARTICLE

Impact of unbalanced minor route versus major route karyotypes at diagnosis on prognosis of CML Alice Fabarius 1 & Lida Kalmanti 1 & Christian T. Dietz 1 & Michael Lauseker 2 & Sébastien Rinaldetti 1 & Claudia Haferlach 3 & Gudrun Göhring 4 & Brigitte Schlegelberger 4 & Martine Jotterand 5 & Benjamin Hanfstein 1 & Wolfgang Seifarth 1 & Mathias Hänel 6 & Claus-Henning Köhne 7 & Hans W. Lindemann 8 & Wolfgang E. Berdel 9 & Peter Staib 10 & Martin C. Müller 1 & Ulrike Proetel 1 & Leopold Balleisen 11 & Maria-Elisabeth Goebeler 12 & Jolanta Dengler 13 & Christiane Falge 14 & Lothar Kanz 15 & Andreas Burchert 16 & Michael Kneba 17 & Frank Stegelmann 18 & Michael Pfreundschuh 19 & Cornelius F. Waller 20 & Karsten Spiekermann 21 & Tim H. Brümmendorf 22 & Matthias Edinger 23 & Wolf-Karsten Hofmann 1 & Markus Pfirrmann 2 & Joerg Hasford 2 & Stefan Krause 24 & Andreas Hochhaus 25 & Susanne Saußele 1 & Rüdiger Hehlmann 1 & for the SAKK and the German CML Study Group

Received: 15 June 2015 / Accepted: 31 August 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Major route additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukaemia (CML) indicate an increased risk of progression and shorter survival. Since major route ACA are almost always unbalanced, it is unclear whether other unbalanced ACA at diagnosis also

confer an unfavourable prognosis. On the basis of 1348 Philadelphia chromosome-positive chronic phase patients of the randomized CML study IV, we examined the impact of unbalanced minor route ACA at diagnosis versus major route ACA on prognosis. At diagnosis, 1175 patients (87.2 %) had

Data presented in part at ASH 2012 and 2013. Alice Fabarius, Lida Kalmanti and Christian T. Dietz contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00277-015-2494-9) contains supplementary material, which is available to authorized users. * Alice Fabarius [email protected] 1

III. Medizinische Universitätsklinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Pettenkoferstrasse 22, 68169 Mannheim, Germany

7

Klinik für Onkologie und Hämatologie, Klinikum Oldenburg, Oldenburg, Germany

8

Klinik für Hämatologie und Onkologie, St.-Marien-Hospital Hagen, Hagen, Germany

9

Medizinische Klinik A, Universitätsklinikum Münster, Münster, Germany

2

Institut für Medizinische Informationsverarbeitung, Biometrie und Epidemiologie (IBE), Ludwig-Maximilians-Universität München, München, Germany

10

Klinik für Hämatologie und Onkologie, St.-Antonius-Hospital Eschweiler, Eschweiler, Germany

3

MLL Münchner Leukämielabor, München, Germany

11

4

Institut für Humangenetik, Medizinische Hochschule Hannover, Hannover, Germany

Abteilung für Hämatologie-Onkologie, Evangelisches Krankenhaus Hamm, Hamm, Germany

12

Service de génétique médicale, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland

Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany

13

Klinik für Innere Medizin III, Klinikum Chemnitz, Chemnitz, Germany

Abteilung Innere Medizin V, Medizinische Klinik, Universitätsklinikum Heidelberg, Heidelberg, Germany

14

Medizinische Klinik 5, Klinikum Nürnberg Nord, Nord, Germany

5

6

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a translocation t(9;22)(q34;q11) and 74 (5.5 %) a variant translocation t(v;22) only, while a loss of the Y chromosome (−Y) was present in addition in 44 (3.3 %), balanced or unbalanced minor route ACA each in 17 (1.3 %) and major route ACA in 21 (1.6 %) cases. Patients with unbalanced minor route ACA had no significantly different cumulative incidences of complete cytogenetic remission or major molecular remission and no significantly different progression-free survival (PFS) or overall survival (OS) than patients with t(9;22), t(v;22), −Y and balanced minor route karyotypes. In contrast, patients with major route ACA had a shorter OS and PFS than all other groups (all pairwise comparisons to each of the other groups: p ≤ 0.015). Five-year survival probabilities were for t(9;22) 91.4 % (95 % CI 89.5–93.1), t(v; 22) 87 % (77.2–94.3), −Y 89.0 % (76.7–97.0), balanced 100 %, unbalanced minor route 92.3 % (72.4–100) and major route 52.2 % (28.2– 75.5). We conclude that only major route, but not balanced or unbalanced minor route ACA at diagnosis, has a negative impact on prognosis of CML. Keywords Chronic myeloid leukaemia . Balanced and unbalanced karyotypes . Cytogenetics . Prognosis . Outcome

Introduction Recently, we have shown that additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukaemia

(CML) impact differently on disease progression. Patients with major route ACA showed shorter overall and progression-free survival (OS, PFS) than minor route ACA and transformed to accelerated phase or blast crisis (AP, BC) more frequently [1]. Our data were in agreement with previous reports showing that patients with ACA have lower cytogenetic response rates under imatinib [2] and that major route ACA and complex karyotypes have the worst outcome of all patients with ACA [3]. Furthermore, it has become generally accepted that acquired genetic instability (independent from time point of occurrence) is a consequence of the reciprocal translocation t(9;22)(q34;q11) [4] resulting in the BCR-ABL1 fusion gene and seems to cause or to promote the occurrence of ACA and mutations during the course of the disease (clonal evolution). ACA are therefore signs of disease progression, rise during the course of untreated disease to more than 80 % in BC and indicate a poor prognosis [5–8]. On the basis of these data, current European LeukemiaNet (ELN) recommendations define major route ACA at diagnosis as a warning sign and newly arising ACA under treatment as failure [9]. Since almost all major route ACA at diagnosis of CML are unbalanced cytogenetic aberrations [1, 7, 8] (except t(3;21)(q26;q22) and inv(3)(q21q26)), it would have been interesting to determine whether other unbalanced aberrations, for instance unbalanced minor route ACA, had a similarly unfavourable impact. Unfortunately, the sample size precluded this analysis at the time. We meanwhile have collected a sufficiently large number of patients to address also this point. The current paper therefore addresses the question whether or not unbalanced minor route ACA exert the same negative impact as major route ACA.

15

Medizinische Klinik, Abteilung II, Universitätsklinikum Tübingen, Tübingen, Germany

16

Klinik für Innere Medizin, Schwerpunkt Hämatologie, Onkologie und Immunologie, Universitätsklinikum Marburg, Marburg, Germany

17

II. Medizinische Klinik und Poliklinik, Universitätsklinikum Schleswig-Holstein, Kiel, Germany

18

Klinik für Innere Medizin III, Universitätsklinikum Ulm, Ulm, Germany

Patients and methods

19

Klinik für Innere Medizin I, Universitätsklinikum des Saarlandes, Homburg, Germany

20

Abteilung Innere Medizin I, Universitätsklinikum Freiburg, Freiburg, Germany

21

Medizinische Klinik und Poliklinik III, Klinikum der Universität München, München, Germany

22

Medizinische Klinik IV, Uniklinik RWTH Aachen, Aachen, Germany

23

Klinik und Poliklinik für Innere Medizin III, Universitätsklinikum Regensburg, Regensburg, Germany

24

Medizinische Klinik 5, Universitätsklinikum Erlangen, Erlangen, Germany

25

Abteilung für Hämatologie/Onkologie, Universitätsklinikum Jena, Jena, Germany

A total of 1551 patients with Philadelphia-(Ph-) and/or BCRABL1-positive CP CML have been randomized from July 2002 until March 2012 to the CML study IV (imatinib 400 mg versus imatinib 800 mg versus imatinib 400 mg in combination with interferon alpha or low-dose cytarabine (Ara-C) versus imatinib 400 mg after failure of interferon alpha) (Fig. 1) [10]. The definitions of CML phases followed the ELN recommendations [9, 11]. The protocol followed the Declaration of Helsinki and was approved by the ethics committee of the Medical Faculty Mannheim at the University Heidelberg, Germany and by local ethics committees of the participating centres. Written informed consent was obtained from all patients prior to entering the study.

Ann Hematol Fig. 1 Overview of patients (flow diagram)

Cytogenetics and multicolor fluorescence in situ hybridization analysis Cytogenetic analyses of at least 20 Giemsa (G)-banded or reverse (R)-banded bone marrow metaphases at diagnosis were interpreted according to the International System for Human Cytogenetic Nomenclature (ISCN 2013) [12, 13]. Cytogenetic analyses were performed every 6 months until confirmed complete cytogenetic remission (CCR) [14]. Patients with cytogenetic aberrations in Ph-negative clones at diagnosis were excluded from this analysis. Patients with constitutional changes were assigned to the group with standard translocation t(9;22)(q34;q11) or variant translocation t(v;22). All variant translocations t(v;22) were confirmed by fluorescence in situ hybridization analysis (FISH) analysis. Karyotypes with major route ACA (cytogenetic aberrations frequently observed in CML, for example, +8, +19, +Ph) show gains and/or losses of chromosomal material and are defined as unbalanced karyotypes. Karyotypes with minor route ACA can show balanced karyotypes without gains and/or losses of chromosomal material (for example, reciprocal translocations) or unbalanced karyotypes (gains and/or losses) with aberrations rarely observed in CML. In patients showing a complex aberrant karyotype, G- or R-banding analysis was combined with multicolor-FISH (m-FISH) analysis according to the manufacturer’s instructions (Metasystems, Altlussheim, Germany) [15]. Cytogenetic remission was defined according to the ELN recommendations [9]. Real-time quantitative PCR Measurement of the BCR-ABL1 fusion transcript was performed by real-time quantitative PCR (RQ-PCR) assay with

hybridization probes using ABL1 for normalization (Light Cycler 1.5, Roche Diagnostics, Mannheim, Germany) [16]. Molecular diagnostics for residual BCR-ABL transcripts were performed every 3 months until confirmed major molecular remission (MMR) and then every 6 months in two standardized and accredited laboratories with defined conversion factors for equivalence of tests (Mannheim and MLL Munich) [14]. Ratios of BCR-ABL1/ABL1 were calculated and expressed according to the international scale (IS) [17]. Statistical analysis PFS was defined as the time from diagnosis until the beginning of AP, BC or death from any cause whatever event came first. For OS, death from any cause was the only event. Probabilities of PFS and OS were calculated by the Kaplan-Meier method and compared by the log-rank test. Patients were censored at the date of last follow-up. Cumulative incidences of CCR and MMR were estimated by the cumulative incidence function considering competing events [18, 19]. Death or progression without prior CCR or MMR, respectively, MMR, was counted as a competing event. For the estimation of cumulative incidences of CCR and MMR, patients were censored at the date of stem cell transplantation (SCT) or at the date of first administration of a second-generation tyrosine kinase inhibitor (TKI; dasatinib, nilotinib, bosutinib). Comparisons of continuous variables (e.g. age) were done with the Mann-Whitney-Wilcoxon test. Prognostic scores (Euro, EUTOS) were calculated using published formulae [20, 21]. p values lower than 5 % were considered significant. Due to the explorative character of this work, no adjustment of p values was done and all p values have to be interpreted

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descriptively. All analyses were performed with the SAS software Version 9.1.3 (SAS Institute) except for the Gray test which was performed with R 3.02.

Results In total, 1348 patients were analysed cytogenetically. The characteristics of all patients and the flow diagram are shown in Table 1 and Fig. 1, respectively. The age distribution between groups was similar except for the minus Y group which had a higher age. As recently shown, a physiological loss of the Y chromosome in elderly men has to be considered in evaluating −Y in CML [1]. A separate group with male patients with the loss of the Y chromosome is advised. Patients of 1175 (87.2 %) had the standard translocation t(9;22)(q34;q11) only, 74 (5.5 %) showed a variant translocation t(v;22) only and 99 (7.3 %) had ACA. Out of these, 44 patients (3.3 %) showed the loss of the Y chromosome (−Y). 55 Patients (4.1 %) had ACA except −Y. Of the latter, 17 (1.3 %) had balanced minor route [reciprocal translocations, e.g. t(1;21); t(2;16); t(3;12)], 17 (1.3 %) unbalanced minor route and 21 (1.6 %) major route karyotypes (trisomy 8, i(17)(q10), trisomy 19, +der(22)t(9;22)(q34;q11)) (Fig. 1). Table 2 shows the karyotypes of all balanced and unbalanced minor route ACA and of all major route ACA (all unbalanced). More patients with major route ACA fall into the high-risk EUTOS and Euro scores (24 and 29 %, respectively), whereas patients with balanced or unbalanced minor route ACA show no higher risk EUTOS/Euro scores (0 and 6 %) (Table 1). In patients #25, #26 and #45, G-banding analysis was confirmed by m-FISH analysis.

Time to response (CCR, MMR) The cumulative incidences of achieving CCR or MMR showed no differences (statistically not significant) comparing the five groups (standard translocation t(9;22) only, variant translocation t(v;22) only, −Y, major route ACA, minor route ACA (balanced and unbalanced) (data not shown)). The curves of major route ACA versus all other groups segregate, but a statistically significant difference could not be ascertained due to insufficient power. Survival (PFS, OS) Pronounced survival differences were observed between unbalanced minor route and major route ACA with regard to PFS and OS. Patients with major route ACA had significantly worse PFS (Fig. 2; p≤0.003 for all pairwise comparisons with each of the other five groups) and significantly worse OS than all the other groups (Fig. 3; p≤0.015 for all pairwise comparisons with each of the other five groups). Five-year survival probabilities were for t(9;22) 91.4 % (95 % CI 89.5–93.1), t(v; 22) 87 % (77.2–94.3), −Y 89.0 % (76.7–97.0), balanced 100 %, unbalanced minor route 92.3 % (72.4–100) and major route 52.2 % (28.2–75.5). Table 3 summarizes the progression and survival status of all 38 patients with unbalanced karyotypes at diagnosis. Whereas no patients progressed out of 16 informative patients with unbalanced minor route ACA (including 3 patients with complex aberrant karyotype), 11 out of 20 patients (including 9 patients with complex aberrant karyotype) progressed and/ or died in the major route ACA group (55 %) (including 4 patients with complex aberrant karyotype). The response levels in the major route ACA group were much lower (only

Table 1 Patients characteristics (age, EUTOS and Euro score, follow-up) in correlation with the cytogenetic subgroups (t(9;22) only, t(v;22) only, balanced minor route ACA, unbalanced minor route ACA, major route ACA and −Y)

Median age (range)

Male (%) EUTOS score (%)

All patients Male Female

Low High Euro score (%) Low Intermediate High Follow-up (median in months)

Unbalanced minor route ACA 17

Major route ACA

−Y

Total

74

Balanced minor route ACA 17

21

44

1348

54 (16–88) 52 (16–88) 55 (17–70) 58 79.5 20.6 28.8 54.8 16.4 67.7

53 (30–76) 47 (30–70) 56 (43–76) 59 100 0 50.0 44.0 6.0 60.5

47 (18–70) 42 (27–49) 49 (18–70) 35 100 0 59.0 41.0 0 68.6

51 (23–73) 45 (23–73) 59 (52–69) 86 76.2 23.8 42.9 28.6 28.6 77.8

62 (24–80) 62 (24–80) 100 90.4 9.6 29.6 54.6 15.9 62.9

53 (16–88) 51 (16–88) 55 (16–83) 60 89.0 11.0 36.2 52.6 11.2 67.4

t(9;22)

t(v;22)

1175 52 (16–87) 50 (16–87) 55 (16–83) 58 89.4 10.6 36.3 53.1 10.6 67.5

Cytogenetic analyses were made and interpreted according to the International System for Human Cytogenetic Nomenclature (ISCN 2009 and 2013) [13, 17] ACA additional cytogenetic aberrations

Ann Hematol Table 2

Cytogenetic data of patients with CML in CP with additional chromosomal aberrations in Ph-positive cells at diagnosis (ACA, except −Y)

No Sex Age at diagnosis (years) ACA in addition to the translocation t(9;22)(q34;q11) or the variant translocation t(v;22) at diagnosis [no. of analysed metaphases] Balanced minor route ACA, n=17 1

M

60

46,XY,t(9;22)(q34;q11),t(15;17)(q22;p11)[8]

2 3

F M

53 48

46,XX,t(3;12)(p14;q23),t(9;22)(q34;q11)[16]/46,XX[4] 46,XY,t(4;6)(q21;p23),t(9;22)(q34;q11)[20]

4

M

47

46,XY,t(9;22)(q34;q11),t(14;17)(p11;p11)[20]

5 6

M F

48 63

46,XY,t(6;9;22)(q23;q34;q11),der(9)t(9;17)(q31;q22),der(17)t(9;17)(q31;q22)[21] 46,XX,t(5;8)(q14;q23),t(9;22)(q34;q11)[19]/46,XX[2]

7 8

F M

52 30

46,XX,t(2;16)(p2?3;p1?3),t(9;22)(q34;q11)[24] 46,XY,der(9)inv(9)(p22q34)t(9;22)(q34;q11),der(22)t(9;22)(q34;q11)[15]

9

F

56

46,XX,t(1;21)(q21;q22),t(9;22)(q34;q11)[20]

10 F 11 M

60 37

46,XX,t(1;9)(q24;q31),t(9;22)(q34;q11)[15]/46,XX[5] 46,XY,t(9;22)(q34;q11),t(15;20)(q13;p12)[20]

12 M 13 M

55 70

46,XY,der(7)t(7;22)(q11;q11),der(9)t(9;22)(q34;q11),der(22)t(7;22)(q11;q11) [11]/46,XY[9] 46,XY,t(9;22)(q34;q11),t(10;22)(q25;q13)[17]

14 F 15 F 16 M

76 43 42

46,XX,t(9;22)(q34;q11)[7]/46,idem,der(19)t(19;?)(p13.3;?)[13] 46,XX,t(7;7)(p22;q22),t(9;22;9)(q34;q11;p24)[16]/46,XX[20] 46,XY,inv(3)(p13q25),t(9;22)(q34;q11)[17]

17 M 36 46,XY,t(9;22)(q34;q11),t(11;19)(q14.1;q13)[16] Unbalanced minor route ACA, n=17 18 F 48 46,XX,der(1)t(1;22)(p36;q11),der(2)t(2;13)(p16;?)dup(2)(q11q31),del(2)(p13),der(22)t(2;22)(p13;q11)[15] 19 F 38 46,XX,del(1)(q21),t(9;22;1)(q34;q11;q21)[20] 20 F 18 46,XX,t(8;9)(q24;p22),der(9)del(9)(q13q22)t(8;9)(q24;p22),der(20)t(9;20)(q22;q13),t(?;9;22)(?;q34;q11) [16]/47,idem,+10[2] 21 F 61 46,XX,t(9;22)(q34;q11)[21]/46,idem,del(6)(q15q23)[4] 22 F 68 46,XX,del(5)(q13q22),t(9;22)(q34;q11)[24] 23 F 49 46,XX,del(3)(p11p21),t(9,22)(q34;q11)[13] 24 M 25 F

44 47

46,XY,t(9;22)(q34;q11)[17]/46,idem,dup(1)(q31q21)[3] 46,XX,t(9;22)(q34;q11)[2]/46,idem,der(7;11)ins(7;11)(p14;p11q25)t(7;11)(p22;p11)[13]

26 M 27 M 28 F

46 27 46

46,XY,der(9)t(9;22)(q34;q11)del(9)(q33q34),del(22)(q11q12),der(22)t(9;22)(q34;q11)[20] 45,XY,t(9;22)(q34;q11),-21[4]/46,XY[16] 46,XX,der(2)t(2;4)(q37;q21),del(4)(q21),t(9;22)(q34;q11)[20]

29 F

56

46,XX,der(9)add(9)(p23)t(9;22)(q34;q11)inv(9)(p23q34),der(22)t(9;22)(q34;q11)[15]

30 M 49 92,XXYY,i(7)(q10)x2,t(9;22)(q34;q11)x2[5]/46,XY[14] 31 M 40 46,XY,del(5)(q11q14),t(9;22)(q34;q11)[19] 32 M 37 45,XY,t(9;22)(q34;q11),der(15)t(15;17)(q10;q10)[10] 33 F 69 45,X,-X,t(9;22)(q34;q11)[20] 34 F 65 46,XX,der(9),der(11),der(15),der(22)[10] Unbalanced major route ACA, n=21 35 M 59 46,XY,der(9)t(9;22)(q34;q11),ider(22)(q10)t(9;22)(q34;q11)[11]/46,XY[14] 36 M 39 48,XY,+8,t(9;22)(q34;q11),i(17)(q10),+der(22)t(9;22)(q34;q11)[10]/46,XY[4] 37 M 72 47,XY,+8,t(9;22)(q34;q11)[20] 38 M 28 50,XY,+8,+8,t(9;22)(q34;q11),i(17)(q10),+19,+der(22)t(9;22)(q34;q11)[6]/46,XX[22] 39 M 53 46,XY,der(9)t(9;22)(q34;q11),idicder(22)(q11)t(9;22)(q34;q11)[22]/46,XY[3] 40 M 40 47,XY,+8,t(9;22)(q34;q11)[19]/46,XY[6] 41 M 41 44,XY,t(9;22)(q34;q11),-14,i(17)(q10),-18[15]/46,XY[11] 42 M 23 47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11)[2]/46,XY[18] 43 F 44 M

59 48

47,XX,+8,t(9;22)(q34;q11)[2]/46,XX[23] 46,XY,t(9;22)(q34;q11)[6]/47,idem,+8[12]/46,XY[2]

Ann Hematol Table 2 (continued) No Sex Age at diagnosis (years) ACA in addition to the translocation t(9;22)(q34;q11) or the variant translocation t(v;22) at diagnosis [no. of analysed metaphases] 45 M

31

46 M

37

46,XY,t(9;22;10)(q34;q11;p15)[13]/55,idem,+3,+8,+12,+13,+14,+18,+19,+21,+ der(22)t(9;22;10)(q34;q11;p15)[7] 46,XY,t(9;22)(q34;q11)[10]/47,idem,+der(22)t(9;22)(q34;q11)[10]

47 M

28

47,XY,t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11)[20]

48 M 49 F

51 52

46,XY,t(9;22)(q34;q11)[8]/47,idem,+8,i(17)(q10)[18] 47,XX,+8,t(9;22)(q34;q11),i(17)(q10)[10]

50 F 51 M

69 24

46,XX,t(9;22)(q34;q11)[9]/46,XX,der(9)t(9;22)(q34;q11),ider(22)(q10)t(9;22)(q34;q11)[13] 46,XY,t(9;22)(q34;q11)[22]/47,idem,+8[3]

52 M

55

46,XY,t(9;22)(q34;q11)[3]/49,idem,+8,+10,+der(22)t(9;22)(q34;q11)[12]

53 M 54 M

73 55

47,XY,+8,t(9;22)(q34;q11)[4]/46,XY[2] 46,XY,t(1;12)(p34;q24),t(1;9;22)(p36;q34;q11)[21]/48,idem,+8,+9[2]

55 M

57

48,XY,+8,t(9;22)(q34;q11),+19[25]

Cytogenetic analyses were made and interpreted according to the International System for Human Cytogenetic Nomenclature (ISCN 2009 and 2013) [16, 13, 9]. Patients #1–17 show a balanced karyotype and patients #18–55 show an unbalanced karyotype Ph Philadelphia chromosome, No number of patients, F female, M male, CP chronic phase, ACA additional cytogenetic aberrations

8 of 21 patients achieved MMR or MR4) than in the unbalanced minor route ACA group (all 16 informative patients achieved MMR or MR4). The percentage of metaphases analysed with ACA did not have a recognizable influence on prognosis. Second-generation TKI (dasatinib, nilotinib) Seven out of 55 patients with ACA at diagnosis were treated with dasatinib or nilotinib. Three out of 17 patients with balanced minor route ACA and 1 out of 17 patients with unbalanced minor route ACA received second-generation TKI and are still alive. Three out of 21 patients with major route ACA received secondgeneration TKI, 2 have died and 1 is alive.

Discussion Our data show that only major route ACA at diagnosis of CML have a negative impact on prognosis. Unbalanced minor route ACA are associated with similar response and survival rates as t(9;22)(q34;q11) alone, variant translocations alone, loss of the Y chromosome or balanced minor route ACA. The association of major route ACA at diagnosis with unfavourable prognosis demonstrates that their occurrence is a non-random modifier of outcome as observed in BC [7]. Minor route ACA, balanced or unbalanced, have no impact on prognosis. They also seem to be random, but transient and may be a consequence of genetic instability of the BCRABL1-positive CML genome [22, 23]. The prevalence of ACA at diagnosis is much lower than that reported for the

later phases of CML, most notably in BC. This could explain why the rarer types of major route aberrations such as +21, + 17, t(3;21)(q26;q22), inv(3)(q21q26) or monosomy 7 were not detected in our cohort [8]. Our results differ from the findings of Luatti et al. [24] who found an impact of ACA on cytogenetic and molecular responses but not on OS and PFS in patients with ACA, when all ACA types were analysed together. They only report on 21 patients and included within the clonal aberrations the −Y which did not show a different prognostic impact than translocation t(9;22) in our series [1]. The proportion of patients with additional cytogenetic findings at diagnosis is 13 % taking all cytogenetic findings together and thus is in the range of published data. Similarly, the relative frequencies of patients with variant translocation, ACA or the lack of the Y chromosome are within the published range [7]. The proportion of patients with major route ACA at diagnosis is small (1.6 %), but their impact on longterm prognosis is evident. In major route karyotypes, trisomy 8 is the most common ACA, mostly in combination with other aberrations, followed by a second Ph, an i(17)(q10), an isoderivative chromosome 22 [ider(22)(q10)t(9;22)(q34;q11)] and trisomy 19. Our findings agree with other reports [8, 25] and support that secondary major route ACA are not random and play a role in disease progression. This analysis complements and extends our earlier observations which demonstrated a negative prognostic impact of major route ACA at diagnosis. We found that unbalanced ACA per se have no negative prognostic impact like the major route constellations [26, 27]. The mechanism by which major route ACA exert their negative impact is unknown. A possible explanation may be

Ann Hematol Fig. 2 Progression-free survival (PFS) in patients with major route unbalanced ACA compared to minor route unbalanced ACA (p= 0.002), balanced ACA (p= 0.003), the t(9;22), −Y and the variant translocations t(v;22) (p<0.001 for each) was calculated by the Kaplan-Meier method and compared by the logrank test. Patients were censored at the date of last follow-up

that major route ACA destabilize the CML genome, thereby causing further genetic instability and progression [27]. Aneuploidy is one of the most frequently observed chromosomal changes in cancer in general and in leukemia in particular. As most genes on aneuploid chromosomes, e.g. trisomy 8 or trisomy 19, are transcribed and potentially translated [28], aneuploidy in CML may create protein imbalances and result in the production of excess free protein subunits or imperfectly assembled protein complexes [29]. As the capacity of aneuploid cells to undergo gene dosage compensation remains controversial, the resulting Bproteotoxic stress^ may explain the altered behaviour of cells in terms of proliferation, cell-cell signalling, differentiation and Fig. 3 Overall survival (OS) in patients with major route unbalanced ACA compared to minor route unbalanced ACA (p= 0.015), balanced ACA (p= 0.003), the t(9;22), −Y and the variant translocations t(v;22) (p<0.001 for each) was calculated by the Kaplan-Meier method and compared by the logrank test. Patients were censored at the last follow-up

apoptosis [30]. Therefore, progenitors featuring unbalanced ACA in vivo may have the capacity to enforce themselves as distinct clones with enhanced malignancy (=clonal evolution). In the worst case, balanced ACA lead to aberrantly expressed fusion genes which apparently do not influence their proximal and distal genetic landscapes to a recognizable extent. As a matter of fact, subclones with balanced ACA in CML are observed mostly sporadically with the possible exemption of the translocation t(3;21)(q26;q22), t(8;21)(q22;q22) and t(15;17)(q22;q21) or the inversion inv(16)(p13q22) and inv(3)(q21q26). The same may be true for unbalanced minor route ACA. Here, no clear correlation with disease progression is recognizable.

Ann Hematol Table 3

Characteristics of patients with CML in CP and with ACA resulting in an unbalanced karyotype

No. of Table 2

% of metaphases with ACA

Patients with unbalanced minor route ACA 18 100

Complex ACA

Best response

Follow-up (years)

Course

Status

Yes

MMR

5.7

No progression

Alive

19

100

MMR

1.9

No progression

Alive

20 21 22 23

100 16 100 100

MMR MR4 MR4 MR4

3.0 6.3 7.8 7.6

No progression No progression No progression No progression

Alive Alive Alive Alive

24

15

MR4

4.7

No progression

Alive

25 26 27 28

87 100 20 100

CHR MR4 MMR MMR

2.3 8.5

Unknown No progression

† Alive

29 30 31 32 33 34

100 100 100 100 100 100

MR4 MR4 MR4 MR4 MMR MMR

6.9 9.3 5.7 5.6 2.2 1.3 2.5 1.8

No progression No progression No progression No progression No progression No progression No progression No progression

Alive Alive Alive Alive Alive Alive Alive Alive

MR4 None MR4 None CCR

7.9 1.7 5.7 6.9 6.5

No progression BC No progression BC No progression

Alive † after SCT for BC † in remission Alive after SCT Alive after SCT for MF

CHR

7.2

No progression

Alive after SCT

MMR CHR MR4 No MMR None

0.4 3.2 7.7 2.7 5.9

BC Progression No progression BC AP

† SCT, † Alive † Alive after SCT

None MR4 MMR MR4 CHR No MMR No CCR MR4 No MMR No MMR

1.3 3.0 3.8 3.5 3.8 0.8 0.7 2.7 2.5 2.5

Imatinib failure CP No progression BC Imatinib failure Imatinib failure Unknown No progression No progression Progression

†, sepsis Alive Alive SCT, † †, aplasia Alive after SCT No information Alive Alive after SCT †

Patients with unbalanced major route ACA 35 44 36 71 37 100 38 21 39 88 40

76

41 42 43 44 45

58 10 8 60 35

46 47 48 49 50 51 52 53 54 55

50 100 69 100 59 12 80 67 8 100

Yes

Yes

Yes Yes

Yes

Yes

Yes Yes

Yes Yes Yes

ACA additional cytogenetic aberrations, SCT stem cell transplantation, BC blast crisis, MR4 deep molecular response, MMR major molecular remission, AP accelerated phase, CCR complete cytogenetic remission, CP chronic phase, CHR complete haematological remission, MF myelofibrosis † death

The prognostic impact of additional cytogenetic findings at diagnosis of CML is high and consideration of their types important. Second-generation TKI do not seem to have an impact. Major route ACA at diagnosis of CML may represent markers of advanced-phase

disease and identify a small group of patients with significantly poorer OS as compared to all other patient groups. Close observation and monitoring are mandatory and early treatment intensification should be considered.

Ann Hematol Acknowledgments The contributions of Gabriele Bartsch, Andrea Elett, Elke Matzat, Uwe Böhm, Sabine Dean, Christine Folz, Michaela Hausmann, Elke Matzat, Regina Pleil-Lösch, Inge Stalljann and all CML trial participants are acknowledged.

9.

10. Authors’ contribution AF, LK, CD, SS and RH had the primary responsibility for the publication. RH, AH, MP and JH contributed to the design of the study. All others contributed to the collection and assembly of data, cytogenetic, molecular and statistical analysis and the interpretation of results. All authors have checked and approved the final version of the manuscript. Compliance with ethical standards Conflict of interest The CML study IV is supported by the Deutsche Krebshilfe (Nr. 106642), Novartis, Nürnberg, Germany, Kompetenznetz für Akute and Chronische Leukämien (BMBF 01GI0270), José-Carreras Leukämiestiftung (DJCLS H09/01f, H06/04v, H03/01) and the European LeukemiaNet (LSHC-CT-2004-503216). Ethics approval The protocol followed the Declaration of Helsinki and was approved by the ethics committee of the Medical Faculty Mannheim at the University Heidelberg, Germany and by local ethics committees of the participating centres.

11.

12.

13.

14.

15. Consent to participate Written informed consent was obtained from all patients prior to entering the study.

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