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Fusion genes involving MEF2D in B-ALL
fusion-positive patients. Mutations of PHF6, recurrent in T-cell acute lymphoblastic leukemia, also showed an unexpectedly high frequency (50%) in these patients. MEF2D fusion-positive patients were older (median age 9 years) with elevated WBC counts (median: 27,300/ml) at presentation and, as a result, were mostly classified as NCI high risk. Although they responded well to steroid treat- ment, MEF2D fusion-positive patients showed a significantly worse outcome, with 53.3% relapse and subsequent death. Stem cell transplantation was ineffective as salvage therapy. Interestingly, relapse was frequently associated with the presence of CDKN2A/CDKN2B gene deletions. Our obser- vations indicate that MEF2D fusions comprise a distinct subgroup of precursor B-cell acute lym- phoblastic leukemia with a characteristic immunophenotype and gene expression signature, associat- ed with distinct clinical features.
Introduction
Precursor B-cell acute lymphoblastic leukemia (B-ALL) is a heterogeneous disease characterized by a variety of genet- ic abnormalities.1 In approximately one-quarter of B-ALL patients, known as the B-other-subgroup, the known major risk-stratifying cytogenetic abnormalities are absent.2 However, recent studies using advanced analytical approaches have described a range of novel genetic sub- groups among B-other-ALL.3-11
The myocyte enhancer factor 2D (MEF2D) gene, located at 1q22, is present among these newly identified rearrange- ments in B-other-ALL.6,7,10,12-14 Seven known fusion partners: B-cell CLL/lymphoma 9 (BCL9, 1q21), heterogeneous nuclear ribonucleoprotein U-like 1 (HNRNPUL1, 19q13.2), deleted in azoospermia-associated protein 1 (DAZAP1, 19p13.3), colony stimulating factor 1 receptor (CSF1R, 5q32), synovial sarcoma translocation, chromosome 18 (SS18, 18q11.2), signal transducer and activator of transcription 6 (STAT6, 12q13.3), and Forkhead Box J2 (FOXJ2, 12p13.31) have been described, mostly among childhood and young adult B-ALL. The MEF2D gene encodes a member of the transcription factor family involved in the control of muscle and neuronal cell differen- tiation and development, which is regulated by class II his- tone deacetylases.15-17 It has been reported that rearrange- ments result in enhanced MEF2D transcriptional activity and lymphoid transformation, thus contributing to the development of a distinct subtype of high-risk leukemia.7,10 However, the true incidence and clinical characteristics, including outcome, of patients with B-ALL harboring MEF2D fusion genes remains unknown.
In this study, we report the detailed analysis of a sub- group of B-ALL with MEF2D fusions within a Japanese pediatric ALL cohort. The heterogeneous nuclear ribonucleopro- tein H1 gene (HNRNPH1), encoding another family member of heterogeneous nuclear ribonucleoprotein,18 was identi- fied as a new fusion partner of the MEF2D gene. Novel immunophenotypic characteristics and accompanying genetic abnormalities as well as distinctive clinical features of B-ALL harboring MEF2D fusions are evaluated and dis- cussed.
Methods
Patient selection and sample preparation
RNA and DNA samples, obtained from pediatric B-ALL patients and stored in the Tokyo Children’s Cancer Study Group (TCCSG) biobank5,11 were used in this study (Online Supplementary Figure S1, Table S1). As indicated in Online Supplementary Table S1, the majority of cases originated from the
TCCSG L04-16 study19 while others, including 2 B-lymphoblas- tic lymphoma (LBL) patients, originated from different cohorts. All investigations were approved by the institutional review boards and informed consent or assent was obtained from par- ents or guardians based on their age and level of understanding, as described previously.5,11 Online Supplementary Figure S1 shows the analysis carried out on each case.
Total RNA and genomic DNA were extracted from bone mar- row or peripheral blood of patients using the miRNeasy Mini Kit and the QIAamp DNA Mini Kit (Qiagen, Inc., Valencia, CA, USA), respectively.
In this paper, B-other-ALL is defined as B-ALL lacking the major risk stratifying genetic abnormalities, including high hyperdiploidy (≥ 51 chromosomes or DNA index ≥ 1.16), low hypodiploidy/near haploidy (≤ 44 chromosomes), fusions of ETV6-RUNX1, TCF3-PBX1, TCF3-HLF, BCR-ABL1, and MLL rearrangements as well as more recently identified genetic abnormalities, including rearrangements of CRLF2 and ZNF384, Ph-like ALL-related tyrosine kinase fusions as well as MEF2D fusions (Online Supplementary Table S1).
Whole transcriptome sequencing and RT-PCR
From previous whole transcriptome sequencing (WTS) of B- other-ALL, we identified cases with ZNF384 fusions11 as well as other abnormalities (Online Supplementary Figure S1, Table S1).19- 21 We re-analyzed remaining 153 WTS data manipulated by “deFuse”,22 an algorithm for gene fusion discovery, and investi- gated the presence of MEF2D fusions. Details of this data analy- sis have been described previously.11 RT-PCR followed by Sanger sequencing was performed to confirm and screen for fusion transcripts, as described previously,5,11 using the primers listed in Online Supplementary Table S2.
Multiplex Ligation-dependent Probe Amplification (MLPA)
MLPA analyses were performed on genomic DNA using two types of SALSA Reference Kits, P335 and P383 (MRC Holland, Amsterdam, the Netherlands), according to the manufacturer's instructions. After separation of amplified products, using the ABI3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), the results were analyzed using Gene Mapper Software (Applied Biosystems) and data, including informative headers, electropherograms, ratio plots, validation boxes, and report tables were obtained. In this study, we present only results of deletions of the exons targeted in these kits.
Whole exome sequencing (WES)
Exome libraries prepared from 100 ng of genomic DNA were sequenced using SBS v.4 reagents with the HiSeq2500 sequenc- ing system. Details of whole exome data analyses have been described previously.11
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