K-Ras Mutations and N-Ras Mutations in Childhood Acute Leukemias with and without MLL Rearrangements.

Abstract

BACKGROUND. Ras mutations are thought to drive proliferation of leukemic cells. We sought to determine the frequency of N-Ras and K-Ras mutations in a large cohort of childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) with special reference to the presence or absence of MLL gene rearrangements.METHODS. Bone marrow samples from 300 children with B-precursor ALL, and 132 children with de novo AML were studied at initial diagnosis. Southern blot analysis was performed to detect MLL rearrangement. RT-PCR was used to detect common MLL fusion transcripts. cDNA panhandle PCR technology was used to identify the infrequent or unknown MLL partner genes. DNA PCR or RT-PCR followed by direct sequencing for each PCR product was performed to detect mutations at codons 12, 13 and 61 of N-Ras and K-Ras genes.RESULTS. Of the 300 patients with B-precursor ALL, 20 had MLL(+), the MLL fusion transcripts included 10 MLL-AF4, 7 MLL-ENL, 2 MLL-AF9, and 1 MLL-AF10, all had pro-B subtype (with CD10 negative). Of the 132 patients with de novo AML, 16 had MLL(+), including 4 MLL-AF9, 5 MLL-AF10, 2 MLL-ENL, one each of MLL-AF1, MLL-AF4, MLL-ELL, MLL-SEPT6 and MLL-PTD. N-Ras mutations were detected in 2 of 20 MLL(+) ALL and in 26 of 274 MLL(−) ALL patients (P=1.000). N-Ras mutations were detected in 2 of 16 MLL(+) AML and in 14 of 116 MLL(−) AML patients (P=1.000). K-Ras mutations were detected in 8 of 20 MLL(+) ALL patients compared with 29 of 280 MLL(−) ALL patients (P=0.001). K-Ras mutations were detected in 2 of 16 MLL(+) AML patients compared with 6 of 114 MLL(−) AML patients (P=0.256). Taken together, 10 of 20 MLL(+) ALL children harbored Ras mutations: 4 Gly12Asp, 2 Gly12Val, 1 Gly12Ala and 1 Gly13Asp for K-Ras, and 2 Gly12Asp for N-Ras mutations. The 4 Ras mutations in MLL(+) AML consisted of one each of Gly12Ala and Gln61Pro for N-Ras and one each of Gly12Ala and Gly13Asp for K-Ras mutations. The frequency of Ras mutations was higher in MLL(+) ALL than that in MLL(+) AML, but the difference did not reach statistical significance (P=0.176). Twenty-six of 274 MLL(−) ALL patients harbored N-Ras mutations: 6 Gly12Asp, one each of Gly12Cys and Gly12Ala, 6 Gly13Asp, 5 Gln61Leu, 3 Gln61Lys, 2 Gln61Arg, 1 Gln61His, and one with both Gly12Asp and Gly13Asp. Four of MLL(−) ALL patients had both N-Ras and K-Ras mutations. Fourteen of 116 MLL(−) AML patients harbored N-Ras mutations: 2 Gly12Asp, 2 Gly12Cys, 1 Gly12Ser, 2 Gly13Cys, one each of Gly13Asp and Gly13Arg, 2 Gln61His, 2 Gln61Leu, and 1 Gln61Arg. Six MLL(−) AML patients had K-Ras mutations: 2 Gly12Cys, and one each of Gly12Ala, Gly12Asp, Gly12Ser and Gly13Asp but no mutation at codon 61. One MLL(−) AML patient had both N-Ras and K-Ras mutations. For B-precursor ALL, the frequency of Ras mutations in MLL(+) patients was significantly higher than that of MLL(−) ALL (50% vs. 18.6%, P=0.002). There was no difference in the frequency of Ras mutations between MLL(+) AML and MLL(−) AML (P=0.485).CONCLUSIONS. Ras mutations were detected in 20.7% of children with B-precursor ALL and in 17.7% of childhood AML. MLL(+) B-precursor ALL was highly associated with Ras mutations (50%), especially with K-Ras mutations (40%), while MLL(+) AML was not.