Leukemia (2007) 21, 725–731 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE

Rare CBFB-MYH11 fusion transcripts in AML with inv(16)/t(16;16) are associated with therapy-related AML M4eo, atypical cytomorphology, atypical immunophenotype, atypical additional chromosomal rearrangements and low white blood cell count: a study on 162 patients

S Schnittger1, U Bacher2, C Haferlach1, W Kern1 and T Haferlach1

1MLL Munich Leukemia Laboratory, Munich, Germany and 2Department of Clinical Chemistry, Ludwig-Maximilians-University, Munich, Germany

The spectrum of CBFB-MYH11 fusion transcripts in acute core-binding factor complex (CBFC).3 Inv(16) in AML is myeloid leukemia (AML) M4eo with inv(16)/t(16;16) is hetero- associated with an overall favorable prognosis; however, up to geneous. Approximately 85% show type A CBFB-MYH11 fusion 7–9 transcripts. In addition, more than 10 different fusion transcripts 30% of patients relapse. On the molecular level, the CBFB- have been reported. The prognostic impact and biological MYH11 fusion transcripts are heterogeneous, dependent on the 2,10–14 background of rare fusion transcripts remain open. In this exons of the CBFB and MYH11 genes that are fused. Of all study, a molecular characterization of CBFB-MYH11 transcripts patients, 85–88% show the transcript type A. Around 5% each in 162 patients with CBFB-MYH11 positive AML at diagnosis was reveal the transcripts D and E. At least eight more different performed. In total, 128 patients (79.0%) showed the fusion fusion transcript types have been reported, some of those in transcript type A, whereas nine different rare CBFB-MYH11 fusion genes were detected in 34 cases (21.0%). Rare fusion single cases only. In addition to complex aberrant karyotypes transcripts were found more frequently in therapy-related AML and MLL/11q23, inv(16) is one of the most common chromo- (P ¼ 0.0106). Numerical gains of the chromosomes 8, 21 and 22 some aberrations in AML after therapy of a preceding were more frequently associated with type A (28.3%) than with malignancy (t-AML).15–20 Dissing et al.15 found inv(16) in 1% rare fusions (12.9%) (P ¼ 0.012). Median white blood cell (WBC) of all cases with t-MDS or t-AML, in most patients in association count was higher in type A (35.4 G/l; range ¼ 1.1–279 G/l) than in to previous therapy with DNA topoisomerase-II-inhibitors. In cases with rare types (7.8 G/l; range ¼ 0.8–148.0 G/l) (Po0.0001). Rare fusion transcripts were correlated with an atypical addition, the authors suggested that rare fusion types might be cytomorphology not primarily suggestive for the FAB subtype more frequent in t-AML with inv(16). However, owing to the M4eo (P ¼ 0.0203). Immunophenotype revealed lower CD2, limited number of rare CBFB-MYH11 fusion types described in CD13, CD33 and CD90 levels than in type A fusion cases the literature, information with respect to the biological and (P ¼ 0.036, 0.002, 0.029 and 0.045, respectively). However, the prognostic implications is missing. It further remains open type of fusion was not an independent prognostic parameter. whether previous chemotherapy influences the type of mole- Leukemia (2007) 21, 725–731. doi:10.1038/sj.leu.2404531; published online 8 February 2007 cular rearrangements. To clarify these questions, we performed Keywords: AML; M4eo; inv(16); rare fusion types; therapy-related a molecular study on 162 patients with CBFB-MYH11 positive AML AML and inv(16)/t(16;16). We further evaluated whether cytomorphology is influenced by the type of the molecular rearrangement. Introduction Patients characteristics Around 7–10% of all patients with de novo acute myeloid leukemia (AML) show a pericentric inversion of chromosome 16 The study was based on 162 patients with AML with inv(16)/ inv(16)(p13q22) or a balanced translocation t(16;16) 1,2 t(16;16) at diagnosis. The cohort consisted of 75 males (46.3%) (p13;q22). Most cases are associated with the typical and 87 females (53.7%). Of these, 138 patients (85.2%) had de morphology of the FAB subtype AML M4eo, which is novo AML and 24 (14.8%) had t-AML. Median age was 50.7 characterized by specific abnormal mostly immature eosino- 3–6 years (range 18.3–82.9 years). All patients were analyzed by phils with large dark granules. In most cases, staining for non- cytomorphology, cytogenetics, interphase fluorescence in situ specific esterase (NSE) is weaker than in the FAB subtype M4 in 3,4 hybridization (FISH) and polymerase chain reaction (PCR) in general. Only rare cases of inv(16) do not show the typical combination.21–25 In most patient treatment followed the aspect of AML M4eo and are diagnosed as other subtypes such AMLCG99 study, which included de novo as well as t-AML.26 as FAB M2, mostly owing to weak NSE activity. On the molecular level, inv(16) is characterized by a reciprocal rearrangement of the CBFB gene on 16q22 Methods and MYH11 on 16p13.1,3 The pathologic fusion gene is supposed to alter transcriptional regulation, which is mediated by the The cytomorphologic diagnosis was performed according to the French–American–British (FAB) classification.6 Cases were classified as typical AML M4eo, if X5% of pathological Correspondence: Dr S Schnittger, MLL Munich Leukemia Laboratory, eosinophils were present.4,5 Cases with an atypical morphology Max-Lebsche-Platz 31, Munich 81377, Germany. E-mail: [email protected] showed o5% mostly o1% pathological eosinophils. Cyto- Received 13 July 2006; revised 31 October 2006; accepted 2 genetic analysis was performed according to standard proto- November 2006; published online 8 February 2007 cols.22 All cases further underwent interphase FISH with Rare CBFB-MYH11 fusion types in t-AML M4eo S Schnittger et al 726 commercially available CBFB probes (Vysis, Bergisch Gladbach, logic diagnosis of AML M4eo even in highly experienced Germany). Isolation of cells, RNA preparation and cDNA laboratories. synthesis were performed as described before.24 PCR for the CBFB-MYH11 fusion transcript was performed as described elsewhere.11,27 Amplification products were analyzed on 1.5% Immunophenotype agarose gels stained with ethidium bromide. Quantification of fusion transcript levels was performed as described previously.24 The immunophenotype was available from 75 patients (62 with Immunophenotyping with multiparameter flow cytometric type A transcripts and 13 with rare fusion transcripts). AML with analysis was performed in 75 of the 162 patients.28 The inv(16)/t(16;16) is associated with aberrant lymphatic co- percentages of positive leukemic cells using isotype controls expression of CD2.29,30 When compared with cases with fusion were determined for each antigen assessed. transcript A, the rare fusion transcript cases showed a Overall survival (OS) and event-free survival (EFS) analyses significantly weaker CD2-expression (P ¼ 0.036). Furthermore, were performed according to Kaplan–Meier. The correlation the myeloid antigens CD13 (P ¼ 0.002) and CD33 (P ¼ 0.029), with other parameters followed Cox regression based on and the progenitor antigen CD90 (P ¼ 0.045) were expressed continuous values. The comparison of survival curves was with lower density in comparison with standard fusion transcript performed using double-sided log rank test. Comparisons of types. dichotomous variables between different groups were per- formed by the use of two-sided Fisher’s exact test. For statistical analysis, SPSS (version 12.4) software (SPSS, Chicago, IL, USA) Cytogenetics was used. Karyotypes were available in 158/162 cases – in 31/34 cases with rare molecular rearrangements and in 127/128 cases with Results type A rearrangements. In 4/162 cases (2.5%), cytogenetic results were hampered owing to a low number of metaphases. Molecular analysis The incidence of the different CBFB-MYH11 fusion types was Of all 162 patients, 128 (79. 0%) showed the fusion transcript A. compared between different cohorts. The main category was Rare fusion transcripts were found in 34/162 patients (21.0%) represented by inv(16)(p13q22) (rare types: 30/31; 96.8%; type most frequently the fusion transcripts D (16/162; 9.9%) and E A: 110/127; 86.6%). Translocations t(16;16)(p13;q22) were (8/162; 4.9%). In addition, rare fusion types were observed in found in 3.2% (1/31) in the patients with rare fusion types and low incidences of 0.6–1.2%: Avar: n ¼ 1; Bvar: n ¼ 1; F: n ¼ 1; in 9.4% (12/127) in patients with type A rearrangements not G: n ¼ 2; H: n ¼ 1; J: n ¼ 2; S/L: n ¼ 2. (Nomenclature of fusion significant (NS)). Other types such as variants of inv(16)/t(16;16) transcripts was performed according to van Dongen et al.12). involving other chromosomes (two cases), cryptic rearrange- ments (two cases) and a deletion in 16q (one case) were not observed in the cohort with rare fusion types (Table 1). Biological parameters and AML history The most frequent additional aberration in AML with inv(16) The cohort with rare fusion transcripts included 14/34 males is numerical gain of chromosome 22 in 35–40% of all cases, (41.2%) and 20/34 females (58.8%), and the cohort with type A followed by numerical gain of the chromosomes 8 or 21.31,32 fusion transcripts 61/128 males (47.7%) and 67/128 females Thus, patients were categorized into three cytogenetic sub- (52.3%). Median age in the patients with rare fusion types was groups: no additional abnormalities; numerical gain of the 53 years (range 27.7–75.8 years) and in type A 50.0 years (range chromosomes 8, 21 or 22; and other additional aberrations 18.3–82.9 years). Thus, sex and age did not differ significantly (Tables 2 and 3). Subgroup 1 without additional abnormalities between both cohorts. In patients with type A fusion transcripts, was observed in 17/31 cases (54.8%) in patients with rare the median white blood cell (WBC) count was 35.4 G/l (range molecular rearrangements in comparison with 79/127 cases 1.1–279 G/l) in contrast to 7.8 G/l (range 0.8–148 G/l) in rare (62.2%) with standard rearrangements type A (n.s.). Typical rearrangements (Po0.0001). Thus, leukocytes were significantly additional abnormalities (subgroup 2) were more frequent in higher in the patients with type A fusions than in the patients type A fusion (35/40; 25.3%) than in rare rearrangements (5/40; with rare rearrangements. This was also confirmed in multi- 16.1%; Po0.001). In contrast, ‘atypical’ additional abnormal- variate analysis taking the history of AML into account. ities (as specified in Table 3) were mainly found in the cohort The frequency of rare rearrangements was compared in t-AML with rare rearrangements (9/31; 29.0%); in contrast, they and in de novo AML. Rare rearrangements were significantly occurred in 11/127 cases (8.7%) only in the cohort with type more frequent in t-AML (10/24; 41.7%) than in de novo AML A fusion (P ¼ 0.012; Table 3). (24/138; 17.4%; P ¼ 0.0106).

Previous malignancies and therapy Morphology Most of the patients with t-AML M4eo were after breast cancer Cytomorphology was compared between patients with rare (13 cases), one of these in combination with ovarian cancer. fusion transcripts and patients with type A rearrangements. Each two cases had previous uterine cancer, non-Hodgkin Atypical morphology not fulfilling either the definition criteria of lymphoma, thyroid cancer and each one case bronchial cancer, the FAB classification (abnormal eosinophils 45%) or the WHO Hodgkin’s disease, Ewing sarcoma, melanoma and testicular definition (‘sometimes o5%’) was found significantly more cancer. With the exception of three patients (after thyroid cancer frequent in the patients with rare fusion transcripts (19/22; and melanoma) who only obtained surgery and radiation, all 86.4%) than in the patients with type A fusion transcripts (11/75; others were treated with combinations of alkylating agents and 14.7%; P ¼ 0.0025). Thus, in many cases with rare fusion topoisomerase-II-inhibitors or anthracyclines (Table 4). Thus, transcripts, cytomorphology alone will not lead to the morpho- there was no correlation of the breakpoints with previous

Leukemia Rare CBFB-MYH11 fusion types in t-AML M4eo S Schnittger et al 727 Table 1 Cytogenetic aberrations in 158 patients with CBFB-MYH11 rearrangements (rare fusion types: n ¼ 31; type A fusions: n ¼ 127)

Rare fusion types Type A fusions P

n % n % inv(16) 30 96.8 110 86.6 NS t(16;16) 1 3.2 12 9.4 NS Othera 0 0.0 5 3.9 NS Total 31 100.0 127 100.0 NS aOther types of aberrations: 1. 47,XX,+8,del(16)(q13) [2], 46,XX [23] 2. 47,XX,+8.ish ins(16)(q22p13)(CBFss+,MYH sp) [14], 46,XX [3] 3. 46,XY,t(7;16)(p11;p13.1) [20] 4. 48,XY,+8,der(16)inv(16)(p13q22)t(16;17)(p13;q12),der(17)t(16;17)(p13;q12),+22 [14], 46,XY [6] 5. 48,XX,+8,+21 [19], 46,XX [1] cryptic CBFB-MYH11-rearrangement.

Table 2 Additional aberrations in 158 patients with AML and CBFB-MYH11 (rare rearrangements: n ¼ 31; type A: n ¼ 127)

No additional aberrations Typical additional aberrationsa Atypical additional aberrationsb P

n % n % n %

Rare rearrangements (n ¼ 31) 17 54.8 5 16.1 9 29.0 0.012 Type A rearrangements (n ¼ 127) 79 62.2 35 27.6 11 8.7 Total (n ¼ 158) 96 60.8 40 25.3 22 13.9 aTypical additional aberrations: +8, +21,+22. bClonal aberrations other than +8, +21, +22 as detailed in Table 3.

Table 3 Atypical additional aberrations (other than +8, +21, or to fusion types, OS showed a trend to be worse in the patients +22) in 128 patients with type A fusion transcripts and in 31 patients with rare types than in those with type A (rare: 1299 days; type with rare fusion transcripts A: not reached) (P ¼ 0.0695), whereas EFS did not differ significantly between both cohorts (Figure 2). However, when Clonal abnormality Type A Rare fusion the history of AML was taken into account, there was no a significant difference with respect to OS or EFS in dependence t(1;19)(q21;p13) F 1 t(4;4)(p10;p10) 1 F on the fusion types. In a multivariate Cox regression analysis del(5)(q13q33),del(12)(p12) F 1a using etiology, age and fusion type as covariates, de novo del(7q) 4 F etiology was the only independent prognostically favorable del(7)(q22q36),t(10;12)(q21;q24) 1 F parameter for EFS. Thus, the unfavorable effect of rare fusion der(7)t(7;8)(q32;q24), +9 1 transcripts suggested by Kaplan–Meier analysis (Figure 2) was +9 2 1 due to the high prevalence of t-AML in the total cohort. dup(9)(p22p24) F 1 +10 F 1 del(11)(p11p15) F 1 +13 F 1a Discussion +14 1 F add(17)(q25),+r,+mar1 1a F a The CBFB-MYH11 fusion in AML with inv(16) is heterogeneous ins(X;7)(q24;q11q32) F 1 3,10–14 a and comprizes more than 10 different fusion transcripts. ÀX F 1 Total 11/127 (8.7%) 9/31 (29.0%) In this study, we did a comprehensive analysis of rare fusion a type AML M4eo in comparison with type A fusions. The Therapy-related cases. incidence of rare fusion transcripts in inv(16)/t(16;16) was 21.0% (34/162) with 9.9% type D and 4.9% type E was similar to data reported previously (19.4%; 7/36 and 32.2%; treatment, which rendered conclusions about possible breakage 10/31).3,10,12 mechanisms impossible. We performed an analysis of the biological parameters of the patients with rare fusion transcripts in comparison with the patients with standard type A rearrangements. Sex and age Prognosis showed no significant differences. Leukocytes were significantly higher in the patients with type A transcripts (Po0.0001). The complete remission rate following induction therapy was However, the percentage of cases with leukocytes 4100 G/l did 64.3% (9/14) in the patients with rare fusion types and 74.2% not differ significantly in both cohorts. Martin et al.33 found a (58/80) in the patients with fusion type A (n.s.). EFS was worse in significantly worse prognosis for the patients with inv(16) and the patients with t-AML (n ¼ 18) in comparison with de novo leukocytes 4100 G/l. However, this may be related mainly to AML (n ¼ 118) (314 versus 1179 days; P ¼ 0.0169). OS did not complications of induction therapy like tumor lysis syndrome differ significantly between both cohorts (Figure 1). With respect rather than to the biology of the disease.34

Leukemia Rare CBFB-MYH11 fusion types in t-AML M4eo S Schnittger et al 728 Table 4 Primary malignancies and therapy in the 24 patients with t-AML M4eo

Primary malignancy Cases Type A fusion transcripts Rare fusion transcripts (n) Cases Radiation Alkylating TopoII Anthracyclines Cases Radiation Alkylating TopoII Anthracyclines (n) agents inhibitor (n) agents inhibitor

Breast cancera 13 8a 781 7535F 5 Lung cancer 1 1 F 11 F 0 FFF F Uterine cancer 2 2 2 FF F 0 FFF F Non Hodgkin’s 20 FFF F 21 2 F 1 lymphoma Hodgkin’s lymphoma 1 1 F 1 F 10FFF F Ewing Sarcoma 1 1 1 1 1 F 0 FFF F Melanoma 1 1 1 FF F 0 FFF Testicular cancer 1 0 FFF F 1 F 11 F Thyroid cancer 2 0 FFF F 22b FF F

Total241410113 8106816 aIn one case breast cancer in combination with ovarian cancer. bRadionucleid therapy.

OS of de novo versus t-AML M4eo EFS of de novo versus t-AML M4eo 1.0 1.0

de novo AML M4eo (n=118)

0.8 0.8 t-AML M4eo (n=18)

0.6 0.6 Survial Survial 0.4 0.4

de novo AML M4eo (n=118) 0.2 0.2 t-AML M4eo (n=18) p=0.1446 p=0.0169 0.0 0.0

0 2000 4000 0 2000 4000 days days

Figure 1 Overall and EFS in therapy related (dotted line) versus de novo AML M4eo (black line).

OS of AML M4eo with "rare" versus type A EFS in AML M4eo with "rare" versus type A CBFB- MYH11 Fusions CBFB- MYH1 Fusions

1.0 1.0

Type A rearrangements (n=110)

Rare rearrangements (n=23) 0.8 0.8

0.6 0.6 Survial

0.4 Survial 0.4

Type A rearrangements (n=110) Rare rearrangements (n=23) 0.2 0.2

p=0.0695 0.0 0.0 p=0.1325

0 2000 4000 0 2000 4000 days days

Figure 2 Overall and EFS in AML M4eo with rare (dotted line) versus type A fusions (black line).

Leukemia Rare CBFB-MYH11 fusion types in t-AML M4eo S Schnittger et al 729 Therapy-related acute leukemia following exposure to topoi- With respect to prognosis, previously two patients with the somerase-II-inhibitors often demonstrate reciprocal transloca- fusion transcripts D and E were reported to show excellent tions involving the MLL gene on 11q23.19,35 A highly specific response to chemotherapy with complete cytogenetic remission double strand break within a 8.3 kb fragment of the MLL gene and survival of 29 and 40 months. Thus, the authors concluded was identified after in vivo and in vitro exposure to epidophyl- that prognosis in inv(16) was probably independent on the type lotoxins in different T- and B-cell malignancies.36,37 The of fusion transcript.13 Others suggested a correlation with breakpoints within the MLL gene vary between de novo and therapy-related AML and unfavorable prognosis.15 However, t-AML.38,39 Within the AML1 locus at 21q22, a reproducible so far a benefit could not be shown for the patients with inv(16)/ induction of a highly specific double-strand DNA cleavage by t(16;16), who were allografted in comparison with the patients topoisomerase inhibitors was described as well.40,41 In contrast receiving chemotherapy alone.43 It remains open whether this is to these findings in MLL and AML1 rearrangements, the true for the patients with rare CBFB-MYH11 transcripts also. In mechanism of breakage in CBFB-MYH11 is unclear. V(D)J our study, rare types showed a trend to shorter OS. However, recombination was suggested at least for the type A variants.42 when the history of AML (de novo versus t-AML) was Studies of genomic breakpoints in therapy-related M4eo or rare considered, prognosis was not independently associated with CBFB-MYH11 fusions have not been published. Like t-AML with the type of rearrangement. On the other hand, in t-AML a t(11q23), t-AML with inv(16) was supposed to be related to significantly shorter EFS was observed, showing that the previous therapy with topoisomerase inhibitors.15 However, prognostic impact of therapy association was stronger than the several inv(16) were also related to previous therapy with influence of the type of transcript. Thus, the shorter OS in the alkylating agents and frequent additional exposition to radia- cohort with rare types was probably because of the higher tion.17 In our study 21/24 cases with t-AML had received proportion of t-AML in this group and there was no clear combinations of alkylating agents, topoisomerase-II-inhibitors, independent prognostic impact of transcript type. However, the or anthracyclines, most of these in addition to radiation. We trend toward doing worse leaves open whether allogeneic could show that independently of the kind of previous therapy, transplantation strategies should be considered in first complete t-AML M4eo was strongly associated with rare CBFB-MYH11 remission. rearrangements. However, there was no obvious correlation to In conclusion, rare CBFB-MYH11 fusion types in AML with certain agents, and thus it was not possible to draw conclusions inv(16)/t(16;16) are correlated with therapy-related AML M4eo, about possible breakage mechanisms. atypical morphology, atypical additional cytogenetic aberra- Dissing et al.15 showed as well that abnormal fusion tions, a different immunophenotype and low peripheral transcripts occurred more frequently in t-AML – three of four leukocytes, and thus define a biological subtype of AML M4eo. patients with t-AML with inv(16) revealed rare fusion transcripts. It thus can be suggested (although the correlation to certain agents was missing) that the molecular mechanisms of the Acknowledgements inv(16) rearrangement differ in de novo AML and in t-AML as it had been shown for MLL and AML1. With respect to This investigation was performed in part in the Laboratory for cytogenetics, type A fusion transcripts were significantly Leukemia Diagnostics; Medical Department III, (Head: Professor associated with numerical gains of the chromosomes 21, Dr W Hiddemann), Ludwig-Maximilians-University, Munich. We 22 and 8. This was confirmed also in multivariate analysis thank all participants of the AMLCG study group for sending bone taking the history of AML into account. In contrast, we found marrow or blood samples to our laboratory for reference diagnosis rare fusion transcripts significantly associated with additional and for submitting clinical data, as part of the patients were chromosomal abnormalities usually atypical for inv(16). This treated within the AMLCG study group. supports the hypothesis that different pathways are involved in leukemogenesis of these different molecular subtypes of AML M4eo. 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