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Fusion genes involving MEF2D in B-ALL
Supplementary Table S3. Five isoforms of MEF2D-BCL9 fusion were identified in 10 patients, including one case in which different isoforms were present together. Most fre- quently, exon 6 of MEF2D was fused in-frame to exon 10 of BCL9 in 5 patients (Figure 2A, Type (a)), in accordance with previous reports.10 Exons 5 and 6 of MEF2D were also fused to exon 9 of BCL9 in 4 and 1 patient, respective- ly (Types (b) and (c)). In addition, we identified two novel breakpoints (Types (d) and (e)). The predicted protein from all fusions retains the DNA-binding MADS domain of the MEF2D protein, while it lacks most of the c-termi- nal portion of MEF2D as well as most of the functional domains of BCL9.
The structure and sequences of MEF2D-HNRNPUL1 are also presented in Figure 1. Distinct from previous reports, only one isoform joining exon 9 of MEF2D to exon 12 of HNPNPUL1 (Type (f)) was observed among our 6 patients. In the case of MEF2D-HNRNPH1, two isoforms, in-frame fusions joining exons 4 and 7 of MEF2D to exon 5 of HNPNPH1 (Type (g) and (h)), respectively, were iden- tified in the same patient.
Immunophenotypic characteristics of B-ALL patients with MEF2D fusions
It has been reported that B-ALL patients with MEF2D fusions show dull or negative expression of CD10 and overexpression of CD38 antigens, based on immunophe- notypic examination.10 Among our MEF2D fusion-positive cases, CD10 expression was lower than that seen in TCF3-PBX1-positive patients, but not B-others-ALL, as shown in Figure 2, Online Supplementary Figure S3 and Table S4. On the other hand, CD38 expression in MEF2D fusion-positive cases was higher than B-other-ALL patients, but not TCF3-PBX1-positive patients. We also observed that expression of the cytoplasmic m chain was higher in MEF2D fusion-positive cases than that in B-
other-ALL patients, with more than 10% of blasts positive for cytoplasmic m in 8/12 patients (ranging from 5.50 to 99.74%, mean: 54.14 ± 41.38%). Moreover, B-ALL patients with MEF2D fusions frequently exhibited aber- rant CD5 expression, with more than 10% of the blasts positive for CD5 in 7/12 patients (ranging from 0.60 to 73.30%, mean: 27.01 ± 26.67%).
Additional genetic abnormalities in MEF2D fusion-positive patients
To elucidate the frequency of additional genetic abnor- malities in B-ALL with MEF2D fusions, we initially per- formed MLPA on 16 DNA samples from patients with MEF2D fusions. As shown in Online Supplementary Table S5, MLPA analysis revealed that deleted or amplified regions of IKZF1, CRLF2, EBF1, BTG1, PHF6, NF1, EZH2, SUZ12, or PTEN were absent from MEF2D fusion-posi- tive cases, although 11/16 exhibited CDKN2A/CDKN2B deletions at a frequency significantly higher than that among the B-other-ALL patients enrolled on L04-16/L06- 16 study (P<0.001, data not shown). Heterozygous dele- tions of LEF1, PAX5, and ETV6 were detected in 1 case each.
To further investigate the presence of additional genetic abnormalities affecting coding sequences in B-ALL with MEF2D fusions, we carried out WES on 3 DNA samples. We identified 16 mutations within genes that had been previously recognized within cancer: ALK, ARFGER3, BRCA1, BRMS1, C8orf4, ITIH1, MAPK13, NCOR2, NLE, NOTCH1, PHF3, PHF6, PHF10, PIK3R5, RB1, and TET1 (Online Supplementary Table S6).
As mutations involving NOTCH1 and PHF6 are known to be recurrent in T-ALL23-26 and NLE encodes the modula- tor of NOTCH1,27 we were encouraged to investigate abnormalities in NOTCH1 signaling pathway genes and other genes reported as targets of recurrent genetic abnor- malities in T-ALL. Thus, we performed RT-PCR and
Table 2. Additional genetic abnormalities of MEF2D fusion-positive cases.
Case Fusion partner
1 BCL9 2 BCL9 3 BCL9 4 BCL9 5 BCL9 6 BCL9 7 BCL9 8 BCL9 9BCL9
11 HNRNPUL1
12 HNRNPUL1
13 HNRNPUL1
14 HNRNPUL1
15 HNRNPUL1
16 HNRNPUL1
17 HNRNPH1
Gene Chromosome
FBXW7 NLE NOTCH1 PHF6 PTEN WT1
4q31.3 17q12 9q34.3 Xq26.2 10q23.31 11p13
WT WT 6764TC,M2255T WT WT WT
WT WT WT WT WT WT 1282CG,Q428E WT WT R15fs WT WT WT WT 6920 A G, Q2307R, S66C WT WT WTWTWTWTWTWT WT WT WT K16I 692C→T,P231L WT
WT WT WT WT WT 1192T→A,C398S WTWTWTWTWTWT WTWTWTD309HWTWT
WT
WT
WT
WT
WT
WT
WT 904 C
WT
WT
WT
WT
WT
WT
G, R302G 1/16
WT ex2 del (no start codon), C82Y WT WT WT ex2 del (no start codon) WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT D309H WT WT WT 955 C→T, R319X WT WT 2/16 8/16 1/16 1/16
Frequency 1/16 ex: exon; WT: wild-type; fs: frame shift; del: deletion.
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