A 62-year-old female patient with the acute type of ATL received five sessions of mLSG-15 therapy combined with mogamulizumab. A dose-intensified chemotherapy called mLSG-15 therapy is commonly used as an initial treatment for aggressive ATL and consists of VCAP (vincristine, cyclophosphamide, doxorubicin, and prednisone), AMP (doxorubicin, ranimustine, and prednisone), and VECP (vindesine, etoposide, carboplatin, and prednisone). She achieved a complete response (CR) from ATL. Three months later, she was referred to our hospital for allogeneic hematopoietic stem cell transplantation (alloHSCT). On admission, she had persistent fever accompanied by repetitive skin rash and arthralgia. The skin rash exhibited patches of 2-3 cm in diameter and sometimes harbored a subcutaneous mass with pain. Interestingly, these skin and joint symptoms always recovered spontaneously in a few days before recurring in different parts of the body. Peripheral blood (PB) examination showed WBC 4.9 x 109/L (neutrophils 32.5%, lymphocytes 15.0%, monocytes 48.5%, myeloblasts 0.0%, abnormal lymphocytes 0.5%, and monocytoid cells 3.5%), Hb 9.6 g/dL, and platelets 87 x 109/L. The absolute monocyte count in PB was 2.4 x 109/L. Monocytosis had been persistently observed, although ATL cells had hardly been detected in PB by either morphological or immunophenotypic analysis. Lactate dehydrogenase increased slightly (LDH: 247 IU/L, normal range 105-211 IU/L). C-reactive protein was highly elevated (CRP: 20.46 mg/dL). The proviral load (PVL) of HTLV-1 was only 0.45%. A systemic computed tomography scan indicated no hepatosplenomegaly or lymphadenopathy or other signs associated with malignant diseases, infectious diseases, or inflammatory diseases. 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)). Screening analysis for the representative 11 leukemic chimera genes including BCR-ABL1 by polymerase chain reaction was negative. Flow cytometric analysis revealed that the monocytes increased in the BM were positive for CD45, CD33, CD4, CD14, and HLA-DR but negative for CD2, CD13, and CD56. 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)). This chromosomal aberration was not observed at the onset of ATL. 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)).
Therefore, the patient was diagnosed with therapy-related myeloid neoplasm (t-MN), which phenotypically resembled chronic myelomonocytic leukemia (CMML)-1. The latent period between the initial therapy and the onset of t-MN was 10 months. On the contrary, ATL was confirmed as CR, in accordance with the response criteria for ATL. Our case did not have clinical evidence of 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)). She underwent alloHSCT but died of viral complications of encephalomyelitis at 4 months after transplantation (7 months after diagnosis of t-MN).
In order to identify the partner gene of MLL gene rearrangement in this case, we performed RNA sequencing analysis. Total RNA was extracted from the patient's BM sample using a QIAGEN RNeasy Mini Kit (QUIAGEN, Venlo, Netherlands). cDNA libraries for next-generation sequencing were constructed from 24 ng of total RNA using a Truseq RNA Access Library Prep Kit (Illumina, San Diego, CA, USA). Each paired-end indexed library was sequenced to a length of 75 nucleotides per mate (2 x 75 bp) on a Nextseq instrument (Illumina). Sequence reads were processed by our in-house Genomon-RNA pipeline (available at http://genomon.readthedocs.org/ja/latest/, http://genomon.hgc.jp/rna/). Fusion transcripts were detected by Genomon-fusion. The Integrative Genomics Viewer (IGV) version 2.3.57 (https://software.broadinstitute.org/software/igv/download) was used to visualize the fusion-sequence. For validation of the fusion transcripts, reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing were performed. Total RNA was extracted from the patient's BM sample using a PAXgene blood RNA kit (QIAGEN) and QIAqube. The RNA was reverse transcribed into cDNA by using SuperScript  IV VILO  Master Mix (Thermo Fisher Scientific, Massachusetts, USA). DNA was amplified and sequenced using Platinum Taq HF (Thermo Fisher Scientific) and the following primers: primer F 5'-GTGTGGGAGATGGGAGGCT-3' from MLL exon 10 and primer R 5'-CCTCCATCTTCACTTCCTGGG-3' from EP300 exon 15.
Next, to explore the molecular mechanisms of leukemogenesis in this case, we performed a targeted deep sequencing analysis. Flow cytometric sorting of the CD14-positive monocytes and CD4-positive T-cells (control 1) from the patient's peripheral blood was performed using a FACS Aria II (BD Biosciences, San Jose, CA). Oral epithelial cells (control 2) were collected via buccal swab from the patient. Targeted sequencing was performed using 20 ng of DNA via the TruSight Myeloid Panel on the MiSeq platform (Illumina), which included the analysis of the following 54 genes related to myeloid malignancies: ABL1, ASXL1, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CBLC, CDKN2A, CEBPA, CSF3R, CUX1, DNMT3A, ETV6/TEL, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KRAS, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, and ZRSR2. T cells and oral epithelial cells served as a germline control in this case. Bioinformatic analysis was performed using standard procedures.
Cytogenetic and FISH analyses revealed that MLL on chromosome 11q23 was involved in gene rearrangement as the result of chromosomal translocation t(11;22)(q23;q13). RNA sequencing analysis followed by RT-PCR validation was performed to identify the partner gene of MLL rearrangement. RNA sequencing analysis detected a chimeric gene, MLL-EP300, formed by the in-frame fusion of MLL exon 10 to EP300 exon 15. The fusion gene was also confirmed by RT-PCR and Sanger sequencing (Figure 2).
To seek the molecular details underlying leukemogenesis in this case, mutation screening analysis with a targeted deep sequencing method was performed. The panel was focused on myelodysplastic syndrome- (MDS-) and acute myeloid leukemia- (AML-) related genes. Among these 54 genes, SRSF2, TET2 and ASXL1 are frequently mutated in CMML patients. However, any additional somatic mutations were not detected in our case (data not shown).
This study was approved by the Institutional Review Board of the Institute of Medical Science, the University of Tokyo, and informed consent was obtained from the patient in accordance with the Declaration of Helsinki.