Identification of a Novel Chromosomal Translocation t(11;16)(q23;q22) Fusing MLL to Enhancer of mRNA Decapping (EDC)-4 in Smoldering Acute Myeloid Leukemia
Authors: Becker Heiko , Kataoka Keisuke , Greve Gabriele , Duque Afonso Jesús , Ma Tobias , Bleul Sabine , Pantic Milena , Schüler Julia , Duyster Justus , Cleary Michael L , Ogawa Seishi , Lübbert Michael 
Published: October 2019

Abstract
Background: Translocations involving the MLL gene located on chromosome 11q23 are usually found in de novo acute myeloid leukemia (AML) and generally confer a dismal prognosis unless the AF9 gene is involved (Döhner et al., Blood 2010;115:453-74). MLL can be fused to multiple different genes, resulting in the large and growing "MLL recombinome" (Meyer et al., Leukemia 2013;27:2165-76). Thus far, only two genes encoding proteins that are part of the mRNA decapping protein complex (i.e., DCP1A, DCPS) have been described as rarely being fused to MLL. Here, we describe an AML with an indolent disease course arising from myelodysplastic syndrome (MDS) that disclosed a unique MLL fusion with another component of the mRNA decapping complex, i.e., EDC4.
Patient and Methods: Briefly, a 55 year-old female patient presented with an MDS [timepoint (t) -1] that within 10 months progressed to an AML with 2% blood and 40% bone marrow myeloblasts (t0). The patient refused treatment beyond supportive care. Six months later, a marked blast expansion of 80% was detectable in the blood (t1). The patient received 5 cycles of decitabine (t2, cycle 5), followed by 3 months of hydroxyurea (t3). Samples were depleted from CD3+ cells via MACS; CD3+ cells served as germline control. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), and the SureSelect Human All Exon v5 kit (Agilent Technologies) was used for exome capturing from gDNA. Next generation sequencing was performed on an Illumina Hiseq 2500. The analyses were performed as previously described (Kataoka et al. Nature Genetics 2015;47:1304-15, Becker et al. Blood 2014;123:1883-6). NOD scid gamma mice were used as hosts for patient derived tumor xenografts (PDX).
Results: Standard metaphase cytogenetics at the diagnosis of AML (t0) revealed a previously undescribed translocation involving the MLL gene, i.e., t(11;16)(q23;q22), as the sole cytogenetic abnormality. The unknown fusion partner on chromosome 16 was identified by RNA-sequencing as the EDC4 gene (a.k.a. Ge-1), which encodes a key scaffold protein of the mRNA decapping complex; the fusion was confirmed by PCR on cDNA. The translocation led to the in-frame fusion of MLL exon 13 to EDC4 exon 6 which was linked by 19 nucleotides from EDC4 intron 5. The predicted amino acid sequence of the linker was ALNTLLR. Further analyses including exome sequencing on the samples collected over the disease course demonstrated STAG2 as a potential founder mutation that was already present during the MDS (t-1) and persisted throughout the disease course at variant allele frequencies (VAFs) of approximately 45-50%. At the time of transformation to AML (t0), the MLL-EDC4 and two RAS mutations (KRAS p.G13D, NRAS p.G12C) were detectable. Towards the terminal phase (t3), the RAS mutations disappeared and a clone that acquired a mutation in the FLT3 tyrosine kinase domain (TKD; p.D835V; VAF 43%) expanded. Primary blasts from the patient engrafted in NOD scid gamma mice and established a stable PDX serial transplantation mouse model used for drug testing.
Conclusions: This report provides the first demonstration of an MLL-EDC4 in-frame fusion, with potential cooperativity with a founder mutation in the STAG2 splicing factor gene during the transformation of MDS to AML and the additional acquisition of a FLT3-TKD mutation during disease progression. RNA sequencing proved to be a very feasible approach to identify novel fusion partners of known oncogenes such as MLL.

Disclosures
Becker: BMS: Honoraria; Novartis: Honoraria. Kataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Schüler:Oncotest GmbH: Employment. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid.