Frequencies and prognostic impact of RAS mutations in MLL-rearranged acute lymphoblastic leukemia in infants

Abstract

Acute lymphoblastic leukemia in infants represents an aggressive malignancy associated with a high incidence (approx. 80%) of translocations involving the Mixed Lineage Leukemia (MLL) gene. Attempts to mimic Mixed Lineage Leukemia fusion driven leukemogenesis in mice raised the question whether these fusion proteins require secondary hits. RAS mutations are suggested as candidates. Earlier results on the incidence of RAS mutations in Mixed Lineage Leukemia-rearranged acute lymphoblastic leukemia are inconclusive. Therefore, we studied frequencies and relation with clinical parameters of RAS mutations in a large cohort of infant acute lymphoblastic leukemia patients. Using conventional sequencing analysis, we screened neuroblastoma RAS viral (v-ras) oncogene homolog gene (NRAS), v-Ki-ras Kirsten rat sarcoma viral oncogene homolog gene (KRAS), and v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) for mutations in a large cohort (n=109) of infant acute lymphoblastic leukemia patients and studied the mutations in relation to several clinical parameters, and in relation to Homeobox gene A9 expression and the presence of ALL1 fused gene 4-Mixed Lineage Leukemia (AF4-MLL). Mutations were detected in approximately 14% of all cases, with a higher frequency of approximately 24% in t(4;11)-positive patients (P=0.04). Furthermore, we identified RAS mutations as an independent predictor (P=0.019) for poor outcome in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia, with a hazard ratio of 3.194 (95% confidence interval (CI):1.211-8.429). Also, RAS-mutated infants have higher white blood cell counts at diagnosis (P=0.013), and are more resistant to glucocorticoids in vitro (P<0.05). Finally, we demonstrate that RAS mutations, and not the lack of Homeobox gene A9 expression nor the expression of AF4-MLL are associated with poor outcome in t(4;11)-rearranged infants. We conclude that the presence of RAS mutations in Mixed Lineage Leukemia-rearranged infant acute lymphoblastic leukemia is an independent predictor for a poor outcome. Therefore, future risk-stratification based on abnormal RAS-pathway activation and RAS-pathway inhibition could be beneficial in RAS-mutated infant acute lymphoblastic leukemia patients.

Figure 1.

RAS mutations. (A) NRAS exon1 condon12 (Gly>Ser) mutation, corresponding with Patient 2, (B) NRAS exon1 condon12 (Gly>Asp) mutation, corresponding with Patient 14, (C) NRAS exon2 condon61 (Gln>Arg) mutation, corresponding with Patient 4, (D) NRAS exon2 condon61 (Gln>Lys) mutation, corresponding with Patient 13, (E) KRAS exon1 codon12 (Gly>Asp) mutation, corresponding with KOPN-8 cell line, (F) KRAS exon1 condon13 (Gly>Asp) mutation, corresponding with Patient 6, (G) KRAS exon1 condon13 (Gly> Asp) mutation, corresponding with Patient 7.

Figure 2.

Drug cytotoxicity of RAS-mutated and non-mutated infant ALL patients. (A) In vitro prednisolone cytotoxicity in MLL-rearranged infant ALL patients, (B) In vitro dexamethasone cytotoxicity in MLL-rearranged infant ALL patients, (C) In vitro prednisolone cytotoxicity in t(4;11)-rearranged infant ALL patients, (D) In vitro dexamethasone cytotoxicity in t(4;11)-rearranged infant ALL patients. Mean in vitro cytotoxicity responses in RAS-mutated and non-mutated infant ALL patients were statistically analyzed using Mann-Whitney U-test. Error bars represent standard error of the mean. Cytotoxicity data for prednisolone and dexamethasone was available for 63 and 44 MLL-rearranged infants ALL patients and 26 and 18 t(4;11)-rearranged infants, respectively.

Figure 3.

Survival of RAS-mutated and non-mutated infant ALL patients. (A) 5-year event-free survival (EFS), (B) 5-year overall survival (OS), (C) 3-year cumulative incidence of relapse (CIR) for RAS-mutated MLL-rearranged infant ALL patients. Survival data were available for 79 of the 91 MLL-rearranged infant ALL cases. (D) 5-year EFS, (E) 5-year OS, (F) 3-year CIR for RAS-mutated t(4;11)-positive infant ALL patients. Survival data were available for 33 of the 38 t(4;11)-positive infant ALL cases.