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. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Br J Haematol. 2014 Mar 25;166(2):254–259. doi: 10.1111/bjh.12852

Acute Myeloid Leukaemia (AML) with t(6;9)(p23;q34) is Associated with Poor Outcome in Childhood AML Regardless of FLT3-ITD Status: A Report from the Children’s Oncology Group

Katherine Tarlock 1,2,#, Todd A Alonzo 3,4,#, Pilar Palomo Moraleda 5, Robert B Gerbing 4, Susana C Raimondi 4,6, Betsy A Hirsch 4,7, Yaddanapudi Ravindranath 4,8, Beverly Lange 4,9, William G Woods 4,10, Alan S Gamis 4,11, Soheil Meshinchi 1,2,4
PMCID: PMC4079767  NIHMSID: NIHMS573614  PMID: 24661089

Abstract

Acute myeloid leukaemia (AML) with t(6;9)(p23;q34) is a rare subtype associated with FLT3-internal tandem duplication (ITD) and poor outcomes. The clinical outcomes of paediatric patients with t(6;9) with and without FLT3-ITD treated on six consecutive cooperative trails were evaluated. In contrast to patients without t(6;9), those with t(6;9) had a significantly lower complete remission rate, higher relapse rate (RR), and poor overall survival (OS). Within t(6;9) patients, those with and without FLT3-ITD had an OS of 40% and 27% respectively (p>0.9), demonstrating that t(6;9) is a high-risk cytogenetic feature in paediatric AML and its clinical impact is independent of the presence of FLT3-ITD.

Keywords: acute myeloid leukaemia, paediatric, t(6;9)(p23;q34), FLT3-ITD, clinical outcome

INTRODUCTION

Translocation t(6;9) is a rare recurring cytogenetic aberration resulting in the formation of a chimeric fusion gene, DEK-NUP214 that occurs in <2% of adult and paediatric acute myeloid leukaemia (AML) cases (Schwartz et al, 1983; Slovak et al, 2006). This translocation creates a nucleoporin fusion protein thought to result in altered nuclear transport of key proteins, although its role in leukaemogenesis is not yet defined. This translocation has also been identified in myelodysplastic syndromes and Ph-negative chronic myeloid leukaemia. In AML, patients positive for the translocation have been reported to have a poor response to induction chemotherapy and high rate of post-remission relapse (Harrison et al, 2010). Although t(6;9) is known to be highly associated with FLT3-internal tandem duplication (ITD), the clinical significance of this association has not been well defined (Meshinchi et al, 2006; Theide et al, 2002). The lack of clarity of the impact of this association raised the question that the adverse outcome in t(6;9) may be due to the presence of FLT3-ITD and not inherent to the DEK-NUP214 translocation. Currently t(6;9) is considered a high-risk cytogenetic abnormality in many adult trials, although some current paediatric trials, including those of theChildren’s Oncology Group (COG), allocate patients with FLT3-ITD to high-risk treatment arms but those with t(6;9) without FLT3-ITD are treated as standard risk. This study defines the prognostic implications t(6;9) in paediatric AML and the prevalence and contribution to clinical outcome of FLT3-ITD in this population.

MATERIALS AND METHODS

Patients and mutation analysis

All paediatric patients with de novo AML enrolled on the previous six phase III paediatric AML clinical trials conducted by COG or its predecessors, Pediatric Oncology Group (POG) or Children’s Cancer Group (CCG) over a period of 22 years (1988-2010) were eligible for this study. 3790 eligible patients enrolled in AML trials CCG-2861, CCG-2891, POG-9421, CCG-2961, COG-AAML03P1 and COG-AAML0531, of which 2839 with available cytogenetic data were included in this study. Study population and treatment regimens have been previously described (Ravindranath et al, 2005; Lange et al, 2005; Woods et al, 1996, Gamis et al, 2010; Cooper et al, 2012). Diagnostic specimens from patients with t(6;9) underwent FLT3 mutational analysis as previously described (Meshinchi et al, 2001).

Statistical methods

Data were analysed from POG-9421 and CCG-2861, -2891, -2961, up to 24 April 2006, 21 September 2001, 14 January 2004 and 6 November 2009, respectively. For patients treated on COG-AAML03P1 and - AAML0531, data were analysed up to 30 September 2012. Significance of differences in proportions was tested using Chi-squared test and Fischer’s exact test for sparse data. The Mann-Whitney test was used to test differences in medians. The Kaplan-Meier method was used to estimate overall survival (OS), event-free survival (EFS) and disease-free survival (DFS) (Kaplan & Meier, 1958). Estimates of relapse risk (RR) were obtained using methods that account for competing events (Kalbfleish & Prentice, 1980). Definitions of OS, EFS, DFS and RR have been previously described (Cooper et al, 2012). The significance of predictor variables was tested with the log-rank statistic for overall OS, EFS and DFS using Gray’s statistic for RR. Estimates were reported with corresponding two standard errors calculated by Greenwood’s formula. Children without an event were censored at date of last known contact. Statistical significance was defined as a P-value < 0.05.

RESULTS AND DISCUSSION

There were 3790 eligible de novo paediatric AML patients, of whom 2839 had known cytogenetic data and 48 cases (1.7%) of t(6;9)(p23;q34) were identified[CCG-2861 (n=2), CCG-2891 (n=5), POG 9421 (n=7), CCG-2961 (n=10), COG-AAML03P1 (n=7) and COG-AAML0531 (n=17)]. We initially evaluated the disease characteristics and clinical impact of t(6;9) in paediatric AML. Comparison of patient demographics and disease characteristics revealed that patients with and without t(6;9) had similar gender distribution, diagnostic white blood cell (WBC) count and diagnostic marrow blast percentage (Table I). However, t(6;9) was highly associated with older age (p<0.001) and French-American-British (FAB) type, with significantly higher association with FAB M2 (p=0.03), and inverse association with FAB M5 (p<0.001). None of the patients with t(6;9) had FAB M0, M5 or M7. Patients with t(6;9) had a worse induction response, with a complete remission (CR) rate of 67% compared to 79% for those without, (p=0.04). Patients with t(6;9) had a worse outcome with an OS from study entry of 39±15% compared to 57±2% for those without (p=0.03, Figure 1A), with a corresponding EFS from study entry of 32±14% and 45±2%, respectively (p=0.2). RR for patients who achieved an initial CR was 64±18% vs. 42±2% (p=0.04) for those with and without t(6;9) with a corresponding DFS from CR of 33±17% and 50±2% (p=0.1, Figure 1B). We found that patients with t(6;9) had similar outcome to patients with the poor prognostic features monosomy 7 or monosomy5/del5q (Figure 1C, D).

Table I.

Patient demographics and disease characteristics

All t(6;9) patients
(n=48)		 All non-t(6;9) patients
(n=2791)		 t(6;9) with FLT3-ITD
(n=24)		 t(6;9) FLT3-ITD
negative
(n=12)
N % N % P N % N % P
Gender
Male 19 40% 1441 52% 0.1 10 42% 4 33% 0.7
Female 29 60% 1350 48% 14 58% 8 67%
Race
White 32 73% 1936 79% 0.3 20 87% 4 40% 0.01
Black 10 23% 320 13% 0.06 2 9% 6 60% 0.004
Asian 2 5% 103 4% 0.7 1 4% 0 0% 1
Other 0 0% 82 3% 0.4 0 0% 0 0% 1
Unknown 4 350 1 2
Ethnicity
Hispanic 5 11% 433 16% 0.4 2 8% 2 20% 0.6
Not Hispanic 41 89% 2289 84% 22 92% 8 80%
Unknown 2 69 0 2
Age (years):
median (range) 		12.6 (2.6 - 20.4) 8.9 (0 - 29.8) <0.001 12.4 (2.6 - 17.3) 12.7 (3.6 - 16.9) 0.8
WBC count (x109 /l):
median (range) 		25.5 (1.7 - 245.7) 22.4 (0 - 827.2) 0.9 51.6 (1.8 - 245.7) 17 (7.1 - 29.8) 0.02
BM Blasts % 60 (25 - 95) 68 (0 - 100) 0.2 70.2 (42 - 95) 47 (25 - 90) 0.006
FAB Classification
M0 0 0% 90 3% 0.4 0 0% 0 0% 1
M1 7 15% 357 14% 0.7 4 17% 1 8% 0.64
M2 19 41% 705 27% 0.03 10 4% 6 50% 0.7
M4 13 28% 598 23% 0.4 8 24% 3 25% 0.7
M5 0 0% 517 20% <0.001 0 0% 0 0% 1
M6 2 4% 52 2% 0.2 0 0% 0 0% 1
M7 0 0% 170 6% 0.1 0 0% 0 0% 1
Other 5 11% 149 6% 0.2 1 4% 2 17% 0.3
Unknown 2 152 1 0
End of course 1
response
CR 32 67% 2162 79% 0.04 11 46% 9 75% 0.1
Not in CR 16 33% 577 21% 13 54% 3 25%
Unevaluable 0 51 0 0
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WBC, white blood cell; BM, bone marrow; FAB, French-American-British; CR, complete remission

Figure 1.

Figure 1

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Clinical outcome of patients with t(6;9) based on the presence or absence of FLT3-ITD: Patients with t(6;9) have a worse overall survival (A) and Relapse Risk (B) in contrast to those without t(6;9). Outcome for patients with t(6;9) is similar to those with high-risk cytogenetics (C and D). In patients with t(6;9) those with and without FLT3-ITD have similar survival and relapse risk (E and F). FLT3-ITD, FLT3 internal tandem duplication; CR, complete remission; CBF AML, core-binding factor acute myeloid leukemia; cyto, cytogenetics;

Of the 48 patients with t(6;9), diagnostic specimens were available for 36 (75%) patients and were tested for FLT3 mutations. FLT3-ITD was identified in 24 out of 36 evaluable patients (67%), and mutations of the activation loop domain of FLT3 (FLT3-ALM) were identified in 2 patients (6%). Patient demographics and disease characteristics were compared in the t(6;9) cohort with and without FLT3-ITD. Among patients with t(6;9), those with FLT3-ITD had a significantly higher median diagnostic WBC count (51.6 × 109/l vs. 17 × 109/l, p=0.02) as well as a higher median marrow blasts percentage (70% vs. 47%, p=0.006). Within the t(6;9) cohort, black race was significantly associated with FLT3-ITD negative status, whereas no association between FLT3-ITD status and racial classification has been demonstrated within the entire study population (Meshinchi et al, 2006). FLT3-ITD status was not associated with FAB type. CR after induction chemotherapy was achieved in 20 of the 36 evaluable patients (56%), and patients with FLT3-ITD had a CR rate of 46% vs. 75% in those without FLT3-ITD (p=0.1). We then examined the impact on clinical outcome for t(6;9) patients based on the presence or absence of FLT3-ITD. There was no difference in 5-year actuarial OS from study entry for t(6;9) patients with and without FLT3-ITD with an OS of 40±20% vs. 27±30% respectively (p>0.9) (Figure 1E). For patients who achieved a remission after induction chemotherapy, RR for those with and without FLT3-ITD was 55±32% vs. 87±31% (p=0.3) (Figure 1F) with a corresponding DFS from remission of 45±30% vs. 13±23% (p=0.3).

As patients in this study were treated over a 22-year period, we examined the impact of treatment era on outcome in t(6;9). Patients with t(6;9) treated on more contemporary studies COG-AAML03P1/0531 (2004-2010) had an EFS of 48% compared to 14% for those treated on CCG-2861/2891 (1989-1992) and 14% for those treated on POG-9421/COG-2961 (p=0.003) (Figure S1A). Closer analysis of t(6;9) patients treated on AAML03P1/0531 demonstrated those with FLT3-ITD had an EFS of 68% compared to 0% in those without FLT3-ITD (p=0.02), suggesting the outcome for those with FLT3-ITD may have improved with contemporary therapies, whereas the outcome for t(6;9) only patients remains dismal (Figure S1B). We initially described the clinical significance of FLT3-ITD in paediatric AML in 2001, and AAML0531 was the first study to allocate patients with high-risk FLT3-ITD to receive haematopoietic stem cell transplantation (HSCT) in first CR. This intervention may have contributed to improvement in outcome in patients with t(6;9) and FLT3-ITD. Prior to the increased use of HSCT in FLT3-ITD, which includes regimens used in the period 1988-2002, EFS for t(6;9) patients was very poor for those with and without FLT3-ITD, at 9% and 0%, respectively (p=0.8) (Figure S1C).

We inquired whether HSCT impacted clinical outcome in patients with t(6;9) and FLT3-ITD. Of the 24 patients identified, 10 received allogeneic HSCT either in first (N=5) or second CR (N=5). The remaining patients (N=14) received conventional chemotherapy. Six of the 10 (60%) HSCT recipients are long-term survivors compared to only 3/14 (21%) chemotherapy-only recipients.

This study provides conclusive data that paediatric AML patients with t(6;9) are at a high risk of relapse and have a poor outcome independent of the presence of FLT3-ITD. Although the presence of FLT3-ITD may provide a therapeutic target for directed therapy, it is unclear if FLT3-targeted interventions will provide survival benefit in t(6;9) patients. In addition, further understanding of the DEK-NUP214 fusion in leukaemogenesis and its potential role in therapeutic resistance may lead to the development of novel and targeted therapeutic strategies in this population. Although the small sample size precludes analysis of statistical significance, it appears that t(6;9) patients have very poor survival when treated with chemotherapy alone compared to HSCT. This supports the data by Ishiyama et al. (2012) that patients with t(6;9) who receive HSCT have a more favourable outcome and this benefit was most significant for patients in CR at the time of transplant. We and others have previously shown that HSCT provides survival benefit to patients with FLT3-ITD (Meshinchi et al, 2006; Bornhäuser et al, 2007) and the previous COG study AAML0531 as well as the current paediatric phase III AML trial COG AALM1031 risk-stratifies patients with high allelic-ratio FLT3-ITD to consolidation with HSCT. The data presented here highlights the high risk nature of patients with t(6;9) regardless of FLT3-ITD status and suggests that t(6;9) patients should be considered at high risk for treatment failure and may benefit from risk-adapted therapy with consolidation HSCT in first CR. Evaluation of treatment strategies that investigate the role of FLT3-targeted therapies as well as those that could target DEK-NUP214 and the resultant altered nuclear transport, as well as further investigation of the role of HSCT for all patients with t(6;9,) are needed to improve outcomes in this high-risk population.

Supplementary Material

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Acknowledgements

The authors are thankful to the COG AML Reference Bank for providing diagnostic specimens. This work was supported by the National Cancer Institute of the National Institutes of Health grants T32 CA009351 (K.T.), R01 CA114563 (S.M.), R21 CA102624 (S.M.), Human Banking Specimen U24 CA114766, and the COG Grant’s Chair U10 CA98543.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Authorship Contribution: K.T. and S.M. designed and performed research, analysed data, and wrote the manuscript; T.A.A. analysed the data, performed statistical analyses and edited the manuscript; R.B.G. performed statistical analyses and edited the manuscript; P.M.M, S.C.R, B.A.H., Y.R., B.L., W.G.W., and A.S.G. performed research and edited the manuscript.

Conflict-of-interest disclosure: The authors have no competing financial interests to declare.

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