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T. Kaburagi et al.
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Figure 2. Gene diagrams depicting RAS pathway mutations in pediatric patients with acute myeloid leukemia. (A) NF1 mutations (NCBI reference sequence; NM_000267); (B) PTPN11 mutations (NCBI reference sequence; NM_002834); (C) CBL mutations (NCBI reference sequence; NM_005188); (D) NRAS mutations (NCBI reference sequence; NM_002524); (E) KRAS mutations (NCBI reference sequence; NM_004985).
NRAS and KRAS mutations were detected in 44 (13.4%) and 12 (3.7%) patients, respectively (Figure 1). All NRAS and KRAS mutations were missense mutations in codon 12, 13, or 61, which are well known hotspots (Figure 2).43 Six patients concomitantly had two missense mutations in NRAS.
Clinical and cytogenetic characteristics of patients with RAS pathway alterations
The clinical characteristics of patients with RAS path- way alterations are summarized in the Online Supplementary Table S4. Patients with RAS pathway alter- ations showed a significantly higher frequency of detec- tion of monosomy 7 as compared to those without RAS pathway alterations (P<0.001). FLT3-ITD and KIT muta- tions were significantly less frequent in patients with RAS pathway alterations (FLT3-ITD, P=0.001; KIT mutations, P=0.004). Age, sex, or relapse rate were not significantly different between patients with or without each specific RAS pathway alteration.
Patients with CBL mutations had significantly higher WBC count at diagnosis (P=0.026; Online Supplementary Table S4; Online Supplementary Figure S3). The frequency of stem cell transplantation (SCT) was significantly higher in patients with PTPN11 mutations (P=0.035), and signifi- cantly lower in patients with NRAS mutations (P=0.022; Figure 1; Online Supplementary Table S4). PTPN11 muta- tions were significantly fewer (P=0.024) in patients with low risk, i.e., core binding factor (CBF)-AML, and NRAS mutations were significantly higher (P=0.017) in these patients (Figure 1; Online Supplementary Table S4). The fre- quency of detection of NF1 alterations was significantly higher in patients with complex karyotype (P=0.031) and MECOM high expression (P=0.013, Figure 1; Online
Supplementary Table S4). PTPN11 mutations were signifi- cantly more frequently detected in patients with mono- somy 7 (P=0.047), RUNX1 mutations (P=0.004), PRDM16 high expression (P=0.002), and MECOM high expression (P=0.004) (Figure 1; Online Supplementary Table S4). NRAS mutations were frequently detected in inv(16)(p13q22)/CBFB-MYH11 (P=0.001) and monosomy 7 (P=0.013). NRAS mutations were also mutually exclu- sive with FLT3-ITD (P=0.005) and KIT mutations (P=0.040) (Figure 1; Online Supplementary Table S4). Although there was no significant difference, three of six patients with CBL mutations were identified in CBF-AML (P=0.411) (Figure 1; Online Supplementary Table S4).
Prognosis of patients with RAS pathway alterations
We analyzed the prognosis of patients with or without RAS pathway alterations using the Kaplan–Meier method (Figure 3; Online Supplementary Figure S4). Despite the small sample size, alterations of NF1 and PTPN11 showed a significant association with poor prognosis. Although there was no significant difference in EFS between patients with or without NF1 alterations, the OS of patients with NF1 alterations was significantly worse than that of patients without NF1 alterations (2- year OS, 42.9% vs. 82.3%, P=0.003) (Figure 3A and B). Although no significant differences were observed in OS, PTPN11 mutations were significantly associated with poor EFS (2-year EFS, 30.0% vs. 59.8%, P=0.013) (Figure 3C and D). The OS and EFS of patients with NRAS muta- tions were significantly better than those of patients without NRAS mutations (2-year OS, 97.7% vs. 79.0%, P=0.014; 2-year EFS, 74.9% vs. 55.9%, P=0.021) (Figure 3E and F). The presence of CBL or KRAS mutations showed no significant impact on prognosis (Online
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