Coexistence and Prognostic Significance of EVI1 Expression and Driver Mutations in KMT2A-Rearranged Acute Myeloid Leukemia
Authors: Matsuo Hidemasa , Yoshida Kenichi , Nakatani Kana , Kamikubo Yasuhiko , Tomizawa Daisuke , Taga Takashi , Kiyokawa Nobutaka , Ogawa Seishi , Meggendorfer Manja , Haferlach Claudia , Adachi Souichi 
Published: November 2019

Background:
KMT2A(MLL)-rearrangements are among the most frequent chromosomal abnormalities in acute myeloid leukemia (AML) and high EVI1 expression is known as an adverse prognostic factor in this subgroup. A recent study showed driver mutations are associated with poor prognosis in pediatric KMT2A-rearranged AML, however, the relationship between EVI1 expression and driver mutations is unclear. In this study, we examined coexistence and prognostic significance of these genetic abnormalities in pediatric and adult KMT2A-rearranged AML.
Patients and Methods:
Forty-four pediatric KMT2A-rearranged AML samples collected in the AML-05 study, conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG), were analyzed for 338 genes by targeted sequencing. In addition, 85 adult KMT2A-rearranged AML samples in MLL Munich Leukemia Laboratory were analyzed for 16 genes (ASXL1, BRAF, CBL, CEBPA, FLT3-ITD, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1, TP53 and WT1) by amplicon deep-sequencing, direct Sanger sequencing, or melting curve analysis. In both studies, EVI1 expression was measured using quantitative RT-PCR.
Results and Discussion:
In pediatric KMT2A-rearranged AML (n=44), among 28 patients with low EVI1 expression, 13 patients (EVI1-WT, 46.4%) had no driver mutations and 15 patients (EVI1-MT, 53.6%) had one or more driver mutations. Among 16 patients with high EVI1 expression, 2 patients (EVI1+WT, 12.5%) had no driver mutations and 14 patients (EVI1+MT, 87.5%) had one or more driver mutations. Mutations in activated signaling pathway genes (FLT3, NRAS, KRAS, PTPN11, CBL, and BRAF) were detected in most patients with EVI1-MT (12/15, 80.0%) and EVI1+MT (13/14, 93.3%). The frequency of mutations in epigenetic regulator genes (SETD2, ASXL1, ASXL2, BCOR, KDM6A, and CREBBP) was significantly higher in EVI1-MT patients (7/15, 46.7%), compared with EVI1+MT patients (1/14, 7.1%) (P=0.04). By contrast, the frequency of mutations in cohesion complex genes (STAG2 and SMC3) was significantly lower in patients with EVI1-MT patients (0/15, 0.0%), compared with EVI1+MT patients (4/14, 28.6%) (P=0.04). The frequency of mutations in transcription factor genes (WT1, MECOM, SPI1, GATA2, and RUNX1) was also lower in EVI1-MT patients (2/15, 13.3%), compared with EVI1+MT patients (5/14, 35.7%) (P=0.21). In adult patients, the frequency of ASXL1 mutations was higher in EVI1-MT patients (3/18, 18.8%), compared with EVI1+MT patients (1/29, 3.4%) (P=0.15) and those of WT1 and RUNX1 mutations were lower in EVI1-MT patients (0/18, 0.0%), compared with EVI1+MT patients (4/29, 13.8%) (P=0.28), which were compatible with the tendency in pediatric KMT2A-rearranged AML.
Next, we examined the prognostic significance of EVI1 expression and driver mutations. Compared with EVI1-WT patients, EVI1-MT patients had lower event-free survival (EFS) (3-years EFS: 84.6% vs. 65.2%, P=0.24). EFS of EVI1+MT patients (3-years EFS: 30.8%) was significantly lower than that of EVI1-WT patients (P=0.001) and EVI1-MT patients (P=0.04). EFS of EVI1+WT patients (3-years EFS: 0.0%) was also lower than that of EVI1-WT patients (P=0.003) and EVI1-MT patients (P=0.09). There was no significant difference in EFS between EVI1+WT and EVI1+MT patients (P=0.88). The results of overall survival (OS) were similar except for EVI1+WT patients (n=2) (3-years OS: EVI1-WT 92.3%, EVI1-MT 70.6%, EVI1+WT 100.0%, EVI1+MT 46.6%). Multivariate analysis including EVI1 expression, driver mutations, age, white blood cell count, and KMT2A-MLLT4 fusion showed EVI1+ is an independent prognostic factor for EFS (hazard ratio (HR): 3.02, 95% confidence interval (CI): 1.08-9.48, P=0.04). There was no prognostic significance in driver mutations (HR:1.24, 95%CI: 0.39-4.74, P=0.73). As a whole, adult patients' survival data were lower, however, the tendency was similar to that of pediatric data (3-years EFS: EVI1-WT 41.7%, EVI1-MT 24.5%, EVI1+WT 9.1%, EVI1+MT 13.5%; 3-years OS: EVI1-WT 55.6%, EVI1-MT 30.7%, EVI1+WT 18.2%, EVI1+MT 36.2%).
These data showed that there is an association between EVI1 expression and the pathway of driver mutations, suggesting these abnormalities may have some cooperative mechanisms in leukemogenesis. Compared with driver mutations, high EVI1 expression may have a stronger impact on poor prognosis in KMT2A-rearranged AML, however, the results should be confirmed in the larger cohort.

Disclosures
Ogawa: RegCell Corporation: Equity Ownership; Kan Research Laboratory, Inc.: Consultancy; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.