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
Authors: Ohki Kentaro , Park Myoung-ja , Sano Hitoshi , Hara Yusuke , Shiba Norio , Tomizawa Daisuke , Taga Takashi , Saito Akiko Moriya , Fujimoto Junichiro , Tawa Akio , Horibe Keizo , Adachi Souichi , Hayashi Yasuhide 
Published: October 2019

Abstract

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.