Low Frequency and Poor Prognosis Of MLL -Partial Tandem Duplications In Pediatric Acute Myeloid Leukemia Using MLPA Method: The Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Trial

Background Mixed-lineage leukemia ( MLL )-partial tandem duplications (PTDs) are found in 3-5% of adult acute myeloid leukemia (AML), and are associated with poor prognosis. Report of the incidence and prognostic relevance of MLL -PTD in pediatric AML is limited and large differences in the frequency have been reported. In pediatric AML cases, a frequency of 10-13% for MLL -PTD was detected using mRNA RT-PCR, whereas a frequency of only 2.5% was detected using multiplex ligation-dependent probe amplification (MLPA). We studied the frequency and prognostic effect of MLL- PTD in pediatric patients with AML treated with JPLSG AML-05 trial (between 2006-2010). Methods MLL -PTD of 331 pediatric de novo AML in the AML-05 trial was analyzed from genomic DNA extracted from their diagnostic bone marrow samples using MLPA analysis. We designed a probe mix for MLPA analysis containing adjacent probes within exon 2-5 and exon 7-13 of the MLL gene for the detection of common and rare type MLL -PTD. Exon 17 of the MLL gene was used as an internal control. We also performed RT-PCR to detect MLL -PTD transcripts to allow comparison with the MLPA results. To assess whether MLL -PTD overlap with known gene abnormalities, such as FLT3 , KIT, and NPM1 mutations , mutational analyses of these genes were also performed in patients in the AML-05 trial. Results MLL -PTD was detected in 9 (2.7%) of 331 patients by MLPA analysis. In 303/331 samples mRNA RT-PCR screening for MLL -PTD was performed, and MLL -PTD was detected in 38 (12.5%). In 9 cases, both MLPA and mRNA-RT-PCR were positive for MLL -PTD. The characteristics of the 9 patients with MLL -PTD using MLPA analysis were below. None of the patients harbouring an MLL -rearrangement, t(8;21) or inv(16) revealed a MLL -PTD. All MLL -PTD cases were found in patients with normal cytogenetics. FLT3 -ITD was present in 4 of 9 patients with MLL -PTD, while none of KIT and NPM1 mutation was detected in MLL -PTD cases. There was a significantly higher frequency of FLT3 -ITD in patients with an MLL -PTD than in those without MLL -PTD (p=0.016). Among these 9 patients, 5 patients were classified as FAB-M5a (p=0.0068), and other 4 patients were classified as FAB-M1, M2, M4 and M6a. The age of patients with MLL -PTD was higher than that of patients without MLL -PTD (median 11.8 years (range; 9-15) and 7.4 years (range; 0-17), respectively; p=0.004). Patients with MLL -PTD tend to have higher white blood cell counts (WBC) at initial diagnosis than those without MLL -PTD (median WBC 6.0×10*9/l (range; 1500-151000) versus 2.2×10*9/l (range; 617-985000a) respectively; p=0.18). All 9 patients with MLL -PTD had events. There was a significantly higher frequency of event including refractory disease, relapse and death in patients with an MLL -PTD than in those without MLL -PTD (p=0.001). Only one of 9 patients was achieved complete remission (CR) after induction therapy (p= 1.1×10 -11 ). Six of 9 patients relapsed, and 5 patients died. Conclusion Using DNA-MLPA as a novel screenings technique, low frequency of MLL-PTD in pediatric AML was found. However, MLL -PTD is highly associated with a poor prognosis in pediatric AML. These data suggest that screening for MLL -PTD in pediatric patients with AML is critical not only for outcome prediction but also for risk-adapted therapy. Disclosures: No relevant conflicts of interest to declare.