PMC6791222_01
Characteristics of MLL-EP300-positive therapy-related myeloid neoplasm in the present case. (a) Bone marrow (BM) showed numerous differentiated monocytes without any increase in the number of myeloblasts or adult T-cell lymphoma/leukemia (ATL) cells (May-Giemsa stain, original magnification, 1,000x). Some monocytes exhibited slightly atypical nuclei. (b) Fluorescence in situ hybridization (FISH) analysis for gene rearrangements involving in the mixed lineage leukemia (MLL) gene at 11q23 was performed. The normal MLL gene exhibited a yellow signal (arrow head), whereas a split MLL gene exhibited as a pair of green and red signals (arrows). The BM sample from the present case showed that 78.5% of the interphase cells were positive for the split MLL gene signals. (c) G-banding of the BM material revealed 46,XX, t(11;22)(q23;q13)[17]/46,XX[3]. Red arrows indicate the abnormal chromosomes involving the translocation. (d) Human T-cell leukemia virus type 1 (HTLV-1) provirus DNA analysis was performed by using inverse polymerase chain reaction. CD14-sorted monocytes (lanes 1, 2) showed no bands. CD3-sorted T-cells (lanes 3, 4) failed to show a visible band with the reproducibility, indicating that the case was negative for a major ATL clone. TL-Om1 was an HTLV-1-infected cell line as positive control (lane 5), and distilled and deionized water (DDW) was used for negative control (lane 6).
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fig1
Bone marrow (BM) examination revealed a slight hypocellularity with a predominance of differentiated monocytes (58% of nuclear cell count) without an increase of blast cells (2%) (Figure 1(a)). Fluorescence in situ hybridization (FISH) analysis showed split mixed lineage leukemia (MLL) gene signals in 78% of the interphase cells (Figure 1(b)) but no rearrangement signals with regard to two genes: platelet-derived growth factor receptor alpha and beta. Karyotyping analysis using the G-band method detected the t(11;22)(q23;q13) translocation as a solo cytogenetic abnormality (Figure 1(c)). Human T-cell leukemia virus type 1 (HTLV-1) provirus DNA analysis (via inverse polymerase chain reaction) of CD14-sorted monocytes revealed no monoclonal integration (Figure 1(d)). ATL disease such as increase of ATL cells in PB and BM, swollen lymph nodes, hepatosplenomegaly, and skin involvement of ATL cells. Additionally, the results from PVL and inverse PCR analysis were also consistent with the conclusion that her ATL had remained in CR (Figure 1(d)).
The structures of MLL, EP300, and MLL-EP300 fusion protein and the sequence of the fusion gene. RNA sequencing and RT-PCR followed by Sanger sequencing demonstrated that MLL exon 10 was fused in-frame to EP300 exon 15. The breakpoint is indicated by the arrow and dashed line. MBD, menin-binding domain; AT, AT hooks; NLS, nuclear localization signal; CxxC, motif recognizing unmethylated CpG dinucleotides; PHD, plant homeodomain fingers; BD, bromodomain; TAD, transactivation domain; SET, H3K4 histone methyltransferase domain; TAZ, transcriptional-adaptor zinc-finger domain; CH, cysteine/histidine-rich regions; KIX, kinase-inducible domain of the CREB-interacting domain; KAT, lysine acetyltransferase domain.
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The fusion gene was also confirmed by RT-PCR and Sanger sequencing (Figure 2).