Clonal Architecture of Relapsed MLL-AF9 Acute Myeloid Leukemia in a Child
Authors: Boutroux Hélène , Hirsch Pierre , Bilhou-Nabera Chrystele , Tang Ruoping , Fava Fanny , Lapillonne Hélène , Leverger Guy , Douay Luc , Delhommeau Francois 
Published: October 2019

Introduction

Acute myeloid leukemia (AML) is an aggressive malignancy caused by the accumulation of multiple oncogenetic mutations occurring in a single lineage of hematopoietic progenitors. AML is rare in children and the mutations found are partially different from those in adults, and for some with a lower frequency. Thus, clonal evolution leading to pediatric AML may be specific, and has not been described yet.

Methods

To define clonal evolution from diagnosis to relapse, we performed whole exome sequencing in matched trio of specimens (diagnosis, germline and relapse) in a 9-years old girl presenting AML FAB M5a with t(9;11)(p22;q23) MLL-AF9 and trisomy 8. At diagnosis, we focused on 3 non-silent somatic mutations candidate for leukemogenesis process, confirmed by Sanger method: EED (R355*), GSDMC (R40*) and ELK1 (3’ UTR). In the same time, we performed cell cultures from bone marrow mononucleated cells at diagnosis. CD34 and CD38 cells were cultured either in liquid long term culture medium (LTC IC) or methylcellulose medium.

Results:

A total of 512 colonies were collecte. Our 3 interest mutations and trisomy 8 were tracked by allele-specific PCR, and MLL rearrangement detected by FISH, individually in 267 from the 512 colonies. Exploitable results were found in 164 colonies. Through these results in the different cell populations, we were able to establish the clonal architecture at diagnosis. MLL-AF9 fusion and EED mutation were found together as the first concomitant occurring events in the leukemic clone. Then genotyping of the colonies demonstrated that ELK1 mutation, GSDMC mutation, and trisomy 8 were successively acquired. Additional later mutations such as ASXL1 (frameshift), PTPN11 (E76K), EMP2 (3’UTR) and DGCR14 (P314S) were detected in the relapse sample.

Discussion

The 3 mutations studied in the colonies may impact the progression of the leukemic clone by dysregulating several cellular pathways and networks. First, EED is an essential non-catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates gene silencing through catalysis of histone H3K27 methylation. PRC2 is known to be enhanced in solid neoplasms such as prostate cancer. On the contrary, in myeloid malignancies and myelodysplasic syndromes, it has been recently demonstrated that mutations involving PRC2 subunits (EED, SUZ12 and EZH1/2) were hypomorphic. These loss-of-functions mutations were responsible for chromatin relaxation and induced transcription of genes promoting self-renewal such as HOXA9. Nevertheless, recent sh-RNA studies in a murine model of MLL-AF9 leukemia demonstrated that residual PRC2 enzymatic activity after EED mutation is needed to unable leukemia growth. These data are coherent with our finding that EED mutation is an early event in leukemogenesis, in cooperation with MLL-AF9 rearrangement. Secondly, ELK1 is targeted by RAS-MAPK pathway, thus its mutation can confer an increased proliferation potential when acquired by the leukemic clone, after its maturation has been blocked and its self-renewal increased through previous MLL rearrangement and EED mutation. Finally, GSDMC may be implicated in monocyte count regulation, and mutated in other neoplasms such as melanoma. As a consequence, it is likely that its mutation occurs lately in the evolution of the monoblastic leukemic clone of our patient. The latest event in the clonal evolution in our patient at diagnosis is the acquisition of trisomy 8.

Conclusion

This study highlights the clonal evolution in one pediatric AML, and paves the way for further studies to better understand clonal evolution in children. Elucidating, the succession and the cooperation between driver and secondary mutations, is important for both understanding leukemogenesis and developing innovative therapeutic agents targeting founding anomalies in the leukemic clone at its most precocious stage. Moreover, discovering clonal architecture also unable to find new minimal residual disease markers to assess the therapeutic response and risk stratification.

Disclosures

No relevant conflicts of interest to declare.