Different Patterns of Cooperating Mutations between De Novo AML Patients with MLL-Partial Tandem Duplication and MLL Translocations.

Two-hit model of leukemogenesis has been proposed for AML. In AML with MLL rearrangements (MLL-R), MLL gene is fused to a variety of partner genes through reciprocal chromosomal translocations (MLL/t11q23), or is rearranged to generate a partial tandem duplication (MLL-PTD). The cooperating mutations of AML with MLL-R have not been systematically analyzed. We aimed to determine the cooperating mutations, including receptor tyrosine kinase (RTK) /Ras signaling pathway, NPM1 and myeloid transcription factors in de novo AML with MLL-R. MLL-R was screened by Southern blot analysis. RT-PCR was used to detect common MLL fusion transcripts. cDNA panhandle PCR was used to identify the infrequent or unknown MLL partner genes. Mutational analysis was peformed by DNA/cDNA PCR-GeneScan analysis for FLT3/ITD, by PCR-RFLP followed by direct sequencing for FLT3/TKD, by DNA/cDNA PCR and direct sequencing for N-Ras, K-Ras, c-KIT, c-FMS, PTPN11, NPM1, AML1 and CEBPα. Of the 131 patients with MLL-R, 77 had MLL-PTD and 54 had MLL/t11q23. None of the 131 patients with MLL-R had c-FMS mutations and c-KIT mutation was present in only one patient with MLL/t11q23. NPM1 mutations occurred in one with MLL-PTD and 2 with MLL/t11q23. The frequencies of other cooperating mutations are shown in Table 1. Taken together, cooperating mutations involving RTK/Ras pathway, NPM1, and/or myeloid transcription factors occurred in 71.4% (55/77) of patients with MLL-PTD and 59.3% (32/54) of patients with MLL/t11q23. In MLL-PTD group, coexistence of two mutations occurred in 23 patients. In MLL/t11q23 group, 6 patients had two mutations. Of the 18 patients with MLL-PTD and AML1 mutations, 8 mutations were located in the Runt homology domain (RHD) and 10 in the non-RHD, 15 were frameshift or nonsense mutations and 3 were missense mutations. Fourteen patients with MLL-PTD and AML1 mutations also had mutations of RTK/Ras singling pathway. Three patients with MLL/t11q23 and AML1 mutations, one in the RHD and 2 in the non-RHD, all were missense mutations. Of the 5 patients with MLL-PTD and CEBPα mutations, 3 harbored FLT3/ITD. Patients with MLL-PTD had a significantly higher frequency of cooperating mutations with myeloid transcription factors than patients with MLL/t11q23 (20/77 vs. 3/54, P=0.002), whereas there was no difference in the frequency of mutations involving RTK/Ras pathway between MLL-PTD and MLL/t11q23 groups (51/77 vs. 29/54, P=0.202). Our results showed that patients with de novo AML and MLL-R had a high frequency of cooperating mutations with RTK/Ras signaling pathway, NPM1 or myeloid transcription factors, and the mutation patterns were different between MLL-PTD and MLL/t11q23 groups.