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

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. 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, and EVI1 is a transcriptional target of MLL oncoproteins. 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. 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. 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. 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. 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. 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 EVI1mRNA 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 over-expressing EVI1: values higher than 0.1 were defined as EVI1 and those lower than 0.1 or undetectable