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The ecotropic viral integration site-1 gene (EVI1) encodes a zinc finger protein that functions as a transcriptional regulator of hematopoietic stem cell self-renewal and long-term multilineage repopulating activity.1,2 The mixed lineage leukemia gene (MLL) rearrangements [i.e. t(11q23)] occur at high frequency in pediatric acute myeloid leukemia (AML) patients with EVI1 overexpression,3 and EVI1 is a transcriptional target of MLL oncoproteins.4 EVI1 overexpression has been reported in up to 10% of patients with AML and is associated with an adverse prognosis. However, the prognostic value of EVI1 overexpression has been studied mostly in adult AML.5–9 Only two studies have examined EVI1 overexpression in pediatric AML, but a detailed analysis according to the type of leukemia was not performed because of the small sample size.3,10  Recent data from an international consortium, including those from our group, suggest that pediatric MLL-rearranged AML can be divided into certain risk groups on the basis of different translocation partners.11 However, clinical outcome data leading to risk stratification of the MLL-rearranged subgroups are still scarce and further investigation is necessary to identify new prognostic factors. Here, we retrospectively examined EVI1 expression levels and clinical outcomes of pediatric MLL-rearranged AML patients treated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study.  After excluding patients with acute promyelocytic leukemia, Down syndrome, secondary AML, myeloid/natural killer cell leukemia and myeloid sarcoma, 485 AML patients were enrolled in the AML-05 study. Overall, 42 patients were excluded, mainly because of misdiagnosis. Details of the treatment schedules and risk stratification were described in previous publication.12 This study was conducted in accordance with the principles set down in the Declaration of Helsinki and was approved by the Ethics Committees of all participating institutions. All patients, or the patients’ parents/guardians, provided written informed consent.  RNA obtained from diagnostic bone marrow samples was used to analyze the expression of EVI1 using a previously established EVI1 quantitative real-time polymerase chain reaction assay that covers the various EVI1 splice variants.7 Event-free survival (EFS) was defined as the time from the diagnosis of AML to the last follow up or the first event (failure to achieve remission, relapse, secondary malignancy, or any cause of death). In this study, most of the events were relapses (n=23) and the rest were deaths with sepsis (n=1) and acute respiratory distress syndrome (n=1). Overall survival (OS) was defined as the time from the diagnosis of AML to any cause of death. All tests were two-tailed and P<0.05 was considered statistically significant.  Among 443 eligible AML patients, 69 were diagnosed as MLL-rearranged AML and diagnostic samples from 50 patients were analyzed for EVI1 mRNA expression. No significant differences in the characteristics and clinical outcomes were observed between these 50 patients and the 19 patients who did not have EVI1 data [EFS (P=0.20), OS (P=0.45)]. EVI1 expression levels were dichotomized based on a cut off of 0.1 relative to SKOV3, an ovarian carcinoma cell line overexpressing EVI1: values higher than 0.1 were defined as EVI1+ and those lower than 0.1 or undetectable were defined as EVI1−, as described in a previous study.7 EVI1+ was present in 18 patients (36%). EVI1 expression levels in different MLL translocation partners relative to that in SKOV3 cells are shown in Online Supplementary Figure S1. The clinical features of EVI1+ and EVI1− patients are summarized in Table 1. EVI1+ patients were significantly older (P=0.03) and had a higher WBC count (P=0.01) at the time of diagnosis than EVI1− patients. Most of the MLL-rearranged AML cases were classified as FAB-M5 or FAB-M4. Specifically, most EVI1− patients (84%) presented with FAB-M5 morphology, which was less frequent in EVI1+ patients (22%), consistent with the findings of a previous study.8 EVI1+ was not correlated with sex or MLL translocation partners. The frequency of FLT3-ITD was significantly higher in EVI1+ patients (P=0.04). We also analyzed CEBPA and NPM1 mutations, which are established favorable prognostic factors; however, none of the patients harbored these mutations, except for one EVI1− patient harboring double CEBPA mutations.    Table 1.  Characteristics of patients categorized according to EVI1 expression status.    Next, clinical outcomes were compared between EVI1+ patients and EVI1− patients (Figure 1). In the MLL-rearranged AML cohort (n=50), EVI1+ patients had a significantly worse EFS than EVI1− patients (P<0.0001) (Figure 1A). However, OS did not differ significantly between the two groups (P=0.054) (Figure 1B). Among several types of MLL-rearrangements, MLL-AF9 was the most common translocation (n=29, 58%) (Table 1). Therefore, clinical outcomes in the cohort of MLL-AF9 positive patients were compared between EVI1+ patients (n=11) and EVI1−patients (n=18). The results showed significant differences in EFS (P<0.0001) and OS (P=0.0008) (Figure 1C and D). By contrast, no differences in EFS (P=0.36) or OS (P=0.57) were observed among patients with MLL-rearranged AML after excluding MLL-AF9 positive patients (Figure 1E and F). The clinical outcomes associated with each type of MLL-rearrangement could not be analyzed because of the small sample size. Multivariate Cox regression analysis, including FLT3-ITD, WBC count, and age identified EVI1+ as the only prognostic factor predicting poor EFS in the total cohort of MLL-rearranged AML (hazard ratio (HR), 4.94; P<0.01) and in the MLL-AF9 positive cohort (HR, 33.81; P<0.01), but not OS (Online Supplementary Table S1).    Figure 1.  Kaplan-Meier survival curves of event-free survival (EFS) and overall survival (OS) from the time of diagnosis according to EVI1 expression status. (A) Kaplan-Meier estimates of EFS in the cohort of MLL-rearranged AML in EVI1+ and EVI1− patients. ...    These results suggest that EVI1 overexpression is an independent adverse prognostic factor because of its association with reduced remission duration in pediatric patients with MLL-rearranged AML, especially in patients harboring MLL-AF9. A recent large study identified several novel prognostic MLL-rearranged subgroups, including a favorable-risk MLL-AF1q positive subgroup and a poor-risk MLL-AF6 positive subgroup.11 However, MLL-AF9 positive patients are categorized as an intermediate risk group, and this subgroup may be dichotomized as a favorable and poor-risk subgroup based on EVI1 expression levels. Pretreatment screening for EVI1 expression should be considered in patients with MLL-rearranged AML to enable better risk assessment and alternative consolidation therapies to be considered. Our results need to be confirmed in larger studies because of the limited case numbers.  From a biological viewpoint, the ‘evil’-like adverse effects of EVI1 in patients with MLL-AF9-positive AML were partially elucidated in a recent study in which EVI1 positive cells harboring MLL-AF9 showed distinct morphological, molecular, and mechanistic differences from EVI1 negative cells.13 Moreover, EVI1 overexpression has been linked to CD52 overexpression, which could be a therapeutic target for monoclonal antibody treatment.14 Further investigation is required to identify novel prognostic factors in the various subgroups of MLL-rearranged AML and to develop therapeutic strategies effective for patients with EVI1 overexpression.

EVI1 overexpression is a poor prognostic factor in pediatric patients with mixed lineage leukemia-AF9 rearranged acute myeloid leukemia