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Cytogenetics and Molecular Genetics

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

Abstract

The spectrum of CBFB-MYH11 fusion transcripts in acute myeloid leukemia (AML) M4eo with inv(16)/t(16;16) is heterogeneous. Approximately 85% show type A CBFB-MYH11 fusion transcripts. In addition, more than 10 different fusion transcripts have been reported. The prognostic impact and biological background of rare fusion transcripts remain open. In this study, a molecular characterization of CBFB-MYH11 transcripts in 162 patients with CBFB-MYH11 positive AML at diagnosis was performed. In total, 128 patients (79.0%) showed the fusion transcript type A, whereas nine different rare CBFB-MYH11 fusion genes were detected in 34 cases (21.0%). Rare fusion transcripts were found more frequently in therapy-related AML (P=0.0106). Numerical gains of the chromosomes 8, 21 and 22 were more frequently associated with type A (28.3%) than with rare fusions (12.9%) (P=0.012). Median white blood cell (WBC) count was higher in type A (35.4 G/l; range=1.1–279 G/l) than in cases with rare types (7.8 G/l; range=0.8–148.0 G/l) (P<0.0001). Rare fusion transcripts were correlated with an atypical cytomorphology not primarily suggestive for the FAB subtype M4eo (P=0.0203). Immunophenotype revealed lower CD2, CD13, CD33 and CD90 levels than in type A fusion cases (P=0.036, 0.002, 0.029 and 0.045, respectively). However, the type of fusion was not an independent prognostic parameter.

Introduction

Around 7–10% of all patients with de novo acute myeloid leukemia (AML) show a pericentric inversion of chromosome 16 inv(16)(p13q22) or a balanced translocation t(16;16)(p13;q22).1, 2 Most cases are associated with the typical morphology of the FAB subtype AML M4eo, which is characterized by specific abnormal mostly immature eosinophils with large dark granules.3, 4, 5, 6 In most cases, staining for non-specific esterase (NSE) is weaker than in the FAB subtype M4 in general.3, 4 Only rare cases of inv(16) do not show the typical aspect of AML M4eo and are diagnosed as other subtypes such 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 and MYH11 on 16p13.1, 3 The pathologic fusion gene is supposed to alter transcriptional regulation, which is mediated by the core-binding factor complex (CBFC).3 Inv(16) in AML is associated with an overall favorable prognosis; however, up to 30% of patients relapse.7, 8, 9 On the molecular level, the CBFB-MYH11 fusion transcripts are heterogeneous, dependent on the exons of the CBFB and MYH11 genes that are fused.2, 10, 11, 12, 13, 14 Of all patients, 85–88% show the transcript type A. Around 5% each reveal the transcripts D and E. At least eight more different fusion transcript types have been reported, some of those in single cases only. In addition to complex aberrant karyotypes and MLL/11q23, inv(16) is one of the most common chromosome aberrations in AML after therapy of a preceding malignancy (t-AML).15, 16, 17, 18, 19, 20 Dissing et al.15 found inv(16) in 1% of all cases with t-MDS or t-AML, in most patients in association to previous therapy with DNA topoisomerase-II-inhibitors. In addition, the authors suggested that rare fusion types might be more frequent in t-AML with inv(16). However, owing to the limited number of rare CBFB-MYH11 fusion types described in the literature, information with respect to the biological and prognostic implications is missing. It further remains open whether previous chemotherapy influences the type of molecular rearrangements. To clarify these questions, we performed a molecular study on 162 patients with CBFB-MYH11 positive AML and inv(16)/t(16;16). We further evaluated whether cytomorphology is influenced by the type of the molecular rearrangement.

Patients characteristics

The study was based on 162 patients with AML with inv(16)/t(16;16) at diagnosis. The cohort consisted of 75 males (46.3%) and 87 females (53.7%). Of these, 138 patients (85.2%) had de novo AML and 24 (14.8%) had t-AML. Median age was 50.7 years (range 18.3–82.9 years). All patients were analyzed by cytomorphology, cytogenetics, interphase fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) in combination.21, 22, 23, 24, 25 In most patient treatment followed the AMLCG99 study, which included de novo as well as t-AML.26

Methods

The cytomorphologic diagnosis was performed according to the French–American–British (FAB) classification.6 Cases were classified as typical AML M4eo, if 5% of pathological eosinophils were present.4, 5 Cases with an atypical morphology showed <5% mostly <1% pathological eosinophils. Cytogenetic analysis was performed according to standard protocols.22 All cases further underwent interphase FISH with commercially available CBFB probes (Vysis, Bergisch Gladbach, Germany). Isolation of cells, RNA preparation and cDNA 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% agarose gels stained with ethidium bromide. Quantification of fusion transcript levels was performed as described previously.24 Immunophenotyping with multiparameter flow cytometric analysis was performed in 75 of the 162 patients.28 The percentages of positive leukemic cells using isotype controls were determined for each antigen assessed.

Overall survival (OS) and event-free survival (EFS) analyses were performed according to Kaplan–Meier. The correlation with other parameters followed Cox regression based on continuous values. The comparison of survival curves was performed using double-sided log rank test. Comparisons of dichotomous variables between different groups were performed by the use of two-sided Fisher's exact test. For statistical analysis, SPSS (version 12.4) software (SPSS, Chicago, IL, USA) was used.

Results

Molecular analysis

Of all 162 patients, 128 (79. 0%) showed the fusion transcript A. Rare fusion transcripts were found in 34/162 patients (21.0%) most frequently the fusion transcripts D (16/162; 9.9%) and E (8/162; 4.9%). In addition, rare fusion types were observed in low incidences of 0.6–1.2%: Avar: n=1; Bvar: n=1; F: n=1; G: n=2; H: n=1; J: n=2; S/L: n=2. (Nomenclature of fusion transcripts was performed according to van Dongen et al.12).

Biological parameters and AML history

The cohort with rare fusion transcripts included 14/34 males (41.2%) and 20/34 females (58.8%), and the cohort with type A fusion transcripts 61/128 males (47.7%) and 67/128 females (52.3%). Median age in the patients with rare fusion types was 53 years (range 27.7–75.8 years) and in type A 50.0 years (range 18.3–82.9 years). Thus, sex and age did not differ significantly between both cohorts. In patients with type A fusion transcripts, the median white blood cell (WBC) count was 35.4 G/l (range 1.1–279 G/l) in contrast to 7.8 G/l (range 0.8–148 G/l) in rare rearrangements (P<0.0001). Thus, leukocytes were significantly higher in the patients with type A fusions than in the patients with rare rearrangements. This was also confirmed in multivariate analysis taking the history of AML into account.

The frequency of rare rearrangements was compared in t-AML and in de novo AML. Rare rearrangements were significantly more frequent in t-AML (10/24; 41.7%) than in de novo AML (24/138; 17.4%; P=0.0106).

Morphology

Cytomorphology was compared between patients with rare fusion transcripts and patients with type A rearrangements. Atypical morphology not fulfilling either the definition criteria of the FAB classification (abnormal eosinophils >5%) or the WHO definition (‘sometimes <5%’) was found significantly more frequent in the patients with rare fusion transcripts (19/22; 86.4%) than in the patients with type A fusion transcripts (11/75; 14.7%; P=0.0025). Thus, in many cases with rare fusion transcripts, cytomorphology alone will not lead to the morphologic diagnosis of AML M4eo even in highly experienced laboratories.

Immunophenotype

The immunophenotype was available from 75 patients (62 with type A transcripts and 13 with rare fusion transcripts). AML with inv(16)/t(16;16) is associated with aberrant lymphatic co-expression of CD2.29, 30 When compared with cases with fusion transcript A, the rare fusion transcript cases showed a significantly weaker CD2-expression (P=0.036). Furthermore, the myeloid antigens CD13 (P=0.002) and CD33 (P=0.029), and the progenitor antigen CD90 (P=0.045) were expressed with lower density in comparison with standard fusion transcript types.

Cytogenetics

Karyotypes were available in 158/162 cases – in 31/34 cases with rare molecular rearrangements and in 127/128 cases with type A rearrangements. In 4/162 cases (2.5%), cytogenetic results were hampered owing to a low number of metaphases.

The incidence of the different CBFB-MYH11 fusion types was compared between different cohorts. The main category was represented by inv(16)(p13q22) (rare types: 30/31; 96.8%; type A: 110/127; 86.6%). Translocations t(16;16)(p13;q22) were found in 3.2% (1/31) in the patients with rare fusion types and in 9.4% (12/127) in patients with type A rearrangements not significant (NS)). Other types such as variants of inv(16)/t(16;16) involving other chromosomes (two cases), cryptic rearrangements (two cases) and a deletion in 16q (one case) were not observed in the cohort with rare fusion types (Table 1).

Table 1 Cytogenetic aberrations in 158 patients with CBFB-MYH11 rearrangements (rare fusion types: n=31; type A fusions: n=127)

The most frequent additional aberration in AML with inv(16) is numerical gain of chromosome 22 in 35–40% of all cases, followed by numerical gain of the chromosomes 8 or 21.31, 32 Thus, patients were categorized into three cytogenetic subgroups: no additional abnormalities; numerical gain of the chromosomes 8, 21 or 22; and other additional aberrations (Tables 2 and 3). Subgroup 1 without additional abnormalities was observed in 17/31 cases (54.8%) in patients with rare molecular rearrangements in comparison with 79/127 cases (62.2%) with standard rearrangements type A (n.s.). Typical additional abnormalities (subgroup 2) were more frequent in type A fusion (35/40; 25.3%) than in rare rearrangements (5/40; 16.1%; P<0.001). In contrast, ‘atypical’ additional abnormalities (as specified in Table 3) were mainly found in the cohort with rare rearrangements (9/31; 29.0%); in contrast, they occurred in 11/127 cases (8.7%) only in the cohort with type A fusion (P=0.012; Table 3).

Table 2 Additional aberrations in 158 patients with AML and CBFB-MYH11 (rare rearrangements: n=31; type A: n=127)
Table 3 Atypical additional aberrations (other than +8, +21, or +22) in 128 patients with type A fusion transcripts and in 31 patients with rare fusion transcripts

Previous malignancies and therapy

Most of the patients with t-AML M4eo were after breast cancer (13 cases), one of these in combination with ovarian cancer. Each two cases had previous uterine cancer, non-Hodgkin lymphoma, thyroid cancer and each one case bronchial cancer, Hodgkin's disease, Ewing sarcoma, melanoma and testicular cancer. With the exception of three patients (after thyroid cancer and melanoma) who only obtained surgery and radiation, all others were treated with combinations of alkylating agents and topoisomerase-II-inhibitors or anthracyclines (Table 4). Thus, there was no correlation of the breakpoints with previous treatment, which rendered conclusions about possible breakage mechanisms impossible.

Table 4 Primary malignancies and therapy in the 24 patients with t-AML M4eo

Prognosis

The complete remission rate following induction therapy was 64.3% (9/14) in the patients with rare fusion types and 74.2% (58/80) in the patients with fusion type A (n.s.). EFS was worse in the patients with t-AML (n=18) in comparison with de novo AML (n=118) (314 versus 1179 days; P=0.0169). OS did not differ significantly between both cohorts (Figure 1). With respect to fusion types, OS showed a trend to be worse in the patients with rare types than in those with type A (rare: 1299 days; type A: not reached) (P=0.0695), whereas EFS did not differ significantly between both cohorts (Figure 2). However, when the history of AML was taken into account, there was no significant difference with respect to OS or EFS in dependence on the fusion types. In a multivariate Cox regression analysis using etiology, age and fusion type as covariates, de novo etiology was the only independent prognostically favorable parameter for EFS. Thus, the unfavorable effect of rare fusion transcripts suggested by Kaplan–Meier analysis (Figure 2) was due to the high prevalence of t-AML in the total cohort.

Figure 1
figure1

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

Figure 2
figure2

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

Discussion

The CBFB-MYH11 fusion in AML with inv(16) is heterogeneous and comprizes more than 10 different fusion transcripts.3, 10, 11, 12, 13, 14 In this study, we did a comprehensive analysis of rare fusion type AML M4eo in comparison with type A fusions. The 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%; 10/31).3, 10, 12

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 showed no significant differences. Leukocytes were significantly higher in the patients with type A transcripts (P<0.0001). However, the percentage of cases with leukocytes >100 G/l did not differ significantly in both cohorts. Martin et al.33 found a significantly worse prognosis for the patients with inv(16) and leukocytes >100 G/l. However, this may be related mainly to complications of induction therapy like tumor lysis syndrome rather than to the biology of the disease.34

Therapy-related acute leukemia following exposure to topoisomerase-II-inhibitors often demonstrate reciprocal translocations involving the MLL gene on 11q23.19, 35 A highly specific double strand break within a 8.3 kb fragment of the MLL gene was identified after in vivo and in vitro exposure to epidophyllotoxins in different T- and B-cell malignancies.36, 37 The breakpoints within the MLL gene vary between de novo and t-AML.38, 39 Within the AML1 locus at 21q22, a reproducible induction of a highly specific double-strand DNA cleavage by topoisomerase inhibitors was described as well.40, 41 In contrast to these findings in MLL and AML1 rearrangements, the mechanism of breakage in CBFB-MYH11 is unclear. V(D)J recombination was suggested at least for the type A variants.42 Studies of genomic breakpoints in therapy-related M4eo or rare CBFB-MYH11 fusions have not been published. Like t-AML with t(11q23), t-AML with inv(16) was supposed to be related to previous therapy with topoisomerase inhibitors.15 However, several inv(16) were also related to previous therapy with alkylating agents and frequent additional exposition to radiation.17 In our study 21/24 cases with t-AML had received combinations of alkylating agents, topoisomerase-II-inhibitors, or anthracyclines, most of these in addition to radiation. We could show that independently of the kind of previous therapy, t-AML M4eo was strongly associated with rare CBFB-MYH11 rearrangements. However, there was no obvious correlation to certain agents, and thus it was not possible to draw conclusions about possible breakage mechanisms.

Dissing et al.15 showed as well that abnormal fusion transcripts occurred more frequently in t-AML – three of four 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 inv(16) rearrangement differ in de novo AML and in t-AML as it had been shown for MLL and AML1. With respect to cytogenetics, type A fusion transcripts were significantly associated with numerical gains of the chromosomes 21, 22 and 8. This was confirmed also in multivariate analysis taking the history of AML into account. In contrast, we found rare fusion transcripts significantly associated with additional chromosomal abnormalities usually atypical for inv(16). This supports the hypothesis that different pathways are involved in leukemogenesis of these different molecular subtypes of AML M4eo.

Cytomorphology further showed a strong correlation of rare fusion transcripts to an atypical ‘non-M4eo-like’ morphology characterized either by the absence of pathological eosinophils or by the rare finding of such cells (<5%). Thus, it can be speculated that different kind of leukemogenic mechanisms seem to underly these different CBFB-MYH11-types and end up in different phenotypes. The resulting rare CBFB-MYH11 fusion proteins, usually proteins with higher molecular weight, may have different function compared with the type A fusion protein. Rare fusion proteins may end up in pathways that do not tend to enrich pathologic eosinophils.

According to the FAB classification, the diagnosis of M4eo is based on the detection of at least 5% of pathologic eosinophils.4, 6 Thus, many cases showing inv(16) but with rare transcripts would not have been diagnosed as FAB M4eo on cytomorphological criteria alone. The frequent atypical morphology in cases with rare CBFB-MYH11 fusion types has implications for the diagnosis of M4eo. As according to the WHO criteria, M4eo is defined by genetics in addition to morphology, PCR for CBFB-MYH11 should be recommended at least in cytogenetically difficult cases even in the absence of typical M4eo morphology.

With respect to prognosis, previously two patients with the fusion transcripts D and E were reported to show excellent response to chemotherapy with complete cytogenetic remission and survival of 29 and 40 months. Thus, the authors concluded that prognosis in inv(16) was probably independent on the type of fusion transcript.13 Others suggested a correlation with therapy-related AML and unfavorable prognosis.15 However, so far a benefit could not be shown for the patients with inv(16)/t(16;16), who were allografted in comparison with the patients receiving chemotherapy alone.43 It remains open whether this is true for the patients with rare CBFB-MYH11 transcripts also. In our study, rare types showed a trend to shorter OS. However, when the history of AML (de novo versus t-AML) was considered, prognosis was not independently associated with the type of rearrangement. On the other hand, in t-AML a significantly shorter EFS was observed, showing that the prognostic impact of therapy association was stronger than the influence of the type of transcript. Thus, the shorter OS in the cohort with rare types was probably because of the higher proportion of t-AML in this group and there was no clear independent prognostic impact of transcript type. However, the trend toward doing worse leaves open whether allogeneic transplantation strategies should be considered in first complete remission.

In conclusion, rare CBFB-MYH11 fusion types in AML with inv(16)/t(16;16) are correlated with therapy-related AML M4eo, atypical morphology, atypical additional cytogenetic aberrations, a different immunophenotype and low peripheral leukocytes, and thus define a biological subtype of AML M4eo.

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Acknowledgements

This investigation was performed in part in the Laboratory for Leukemia Diagnostics; Medical Department III, (Head: Professor Dr W Hiddemann), Ludwig-Maximilians-University, Munich. We thank all participants of the AMLCG study group for sending bone marrow or blood samples to our laboratory for reference diagnosis and for submitting clinical data, as part of the patients were treated within the AMLCG study group.

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Correspondence to S Schnittger.

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Schnittger, S., Bacher, U., Haferlach, C. et al. 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. Leukemia 21, 725–731 (2007). https://doi.org/10.1038/sj.leu.2404531

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Keywords

  • AML
  • M4eo
  • inv(16)
  • rare fusion types
  • therapy-related AML

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