The Partial Duplication of the 5′ Segment of KMT2A Revealed KMT2A-MLLT10 Rearrangement in a Boy with Acute Myeloid Leukemia

The duplication of 5′ segment of KMT2A is a rare molecular event in childhood leukemia, and the influence on prognosis is unknown. Here, we report on a boy who developed acute monocytic leukemia. Fluorescence in situ hybridization revealed the duplication of the 5′ segment with 2 normal alleles at KMT2A which was eventually found to be fused with MLLT10. Chemotherapy promptly induced the first complete remission in the patient at our facility, and the patient remained in first complete remission with negative minimal residual disease at 3.5 years from diagnosis. Our case is similar to two previously reported patients who had partial duplication of the 5′ segment of KMT2A with a KMT2A-MLLT10 rearrangement. Further studies and experience with this cryptic translocation may shed more light on the management of acute myeloid leukemia.


Introduction
e histone-lysine N-methyltransferase 2A enzyme (MLL1), encoded by the KMT2A gene, is an upregulator of global, hematopoietic gene transcription, and translocation rearrangement within KMT2A causes variable risk strati cation in acute leukemia based on the nal genetic outcome.
Patients with MLL rearrangement other than t(9;11) and t(11;19) have an inferior outcome [1], and there are additional aberrations in KMT2A rearrangement, such as fusion with preferential partner gene MLLT10, that also carry prognostic signi cance [2].
One of these aberrations is the duplication/ampli cation of the 5′ segment of KMT2A, which is a very rare molecular event, and the in uence of this on patient prognosis is unknown. However, in general, KMT2A ampli cation as an acquired genetic aberration has been reported to result in a poor prognosis [3]. With this deleterious e ect of KMT2A overexpression in acute leukemia in mind, it is possible that even partial ampli cation could a ect the patient's prognosis. In the literature, only 2 cases have been reported: one pediatric case of AML-M5b(FAB: French-American-British Classi cation) with the duplicated 5′ segment of KMT2A relapsed 16 months after diagnosis during maintenance therapy and was later salvaged by allogeneic transplantation [4] and an adult case of AML-M5a(FAB) with an ampli ed 5′ part of 11q23.3 where KMT2A was located eventually needed transplantation [5]. In both literature cases, this partial duplication was paired with KMT2A-MLLT10 rearrangement.
Although preferential fusion with MLLT10 has been well documented, the isolated prognostic importance of the partial ampli cation of KMT2A remains unknown. Here, we present the case of a pediatric male patient with AML who was successfully treated by multiagent chemotherapy alone. e 5′ duplication of KMT2A was identi ed by uorescence in situ hybridization (FISH) before treatment, but fusion to MLLT10 was discovered by RNA sequencing after completion of the treatment even though reverse transcription-PCR at the diagnosis did not detect any fusion partners.
RNA sequencing on NextSeq500 (Illumina, Inc., CA, USA) was then used to screen for fusion partners, revealing a KMT2A-MLLT10 rearrangement. e RNA analysis was conducted as follows: RNA was puri ed from the patient's bone marrow at diagnosis using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. A sequencing library was then generated from 500 ng of total RNA using a TruSeq Stranded mRNA Library Prep Kit v2 (Illumina) according to the manufacturer's instructions. Next, sequencing was conducted at i-Laboratory LLP (Ibaraki, Japan). Obtained reads were aligned   Case Reports in Pediatrics toward human genome assembly hg19, and fusion gene analysis was conducted with CLC Genomics Workbench Ver. 7.5.1 software (Qiagen, Venlo, Netherlands). Four sequencing tags supporting KMT2A-MLLT10 rearrangement were obtained, which were then con rmed by Sanger sequencing. All sequencing data are shown in Table 1. Designed primers were 5′-TCAATTGCTGGCTCAGAAGA-3′ (KMT2A exon 5) and 5′-CTGAGCTATAAGAGCTGCCATT-3′ (MLLT10 exon 16). e KMT2A breakpoint was located on intron 6, which was the upstream region of the sequencing primer for screening at diagnosis, and the MLLT10 breakpoint was located on exon 14. Minimal residual disease (MRD) by reverse transcription-polymerase chain reaction (RT-PCR) was assessed from diagnosis as shown in Figure 1. Although MRD was positive at diagnosis, it changed to negative after induction therapy 1.

Discussion
Pediatric acute myeloid leukemia is classi ed by chromosomal and/or genetic abnormalities according to the World Health Organization Classi cation published in 2008. Chimeric genes including KMT2A rearrangements are used to predict disease outcome. KMT2A rearrangements are seen in about 20% of pediatric AML and are associated with poor outcome, while the disease outcome depends on its partner gene [1]. KMT2A has many partner genes, and each chimeric gene has a di erent prognosis. It is di cult to quantify the e ect of the partial duplication/ampli cation of KMT2A as the two patients previously reported had the same rearrangement of KMT2A-MLLT10 with di erent treatment outcomes. A single abnormality within ampli cation of KMT2A is reported to have a gain-of-function e ect for leukemogenesis [3]; however, the exact role of this "partial" duplication of KMT2A in acute leukemia is still unclear.
It is interesting to note that both previously reported and the current cases have the same rearrangement along with this partial duplication/ampli cation and diagnosis of FAB M5. Hence, we hypothesize that the leukemic cells that partially duplicate KMT2A tend to undergo KMT2A-MLLT10 fusion and may act more similarly to KMT2A-MLLT10 rearrangements caused by the insertion of KMT2A in chromosome 10p or an unbalanced translocation.
is points to di erent causes producing the same prognostic e ect. Similar rearrangement and the duplication of the 5′ segment of KMT2A might result in cell culture di culties in other cases. erefore, we can recommend using FISH assays to detect partial KMT2A duplication, and RNA sequencing may be useful to specify the fusion partner in such cases.

Conclusion
Partial duplication of the 5′ segment of KMT2A can be easily detected by FISH, but the crucial details of the KMT2A-MLLT10 rearrangement may remain hidden from standard PCR testing, which might result in poor prognosis.

Ethical Approval
is study was approved by the ethics committee of the University of Tsukuba Hospital (H23-128) following the Ethical Guidelines for Medical and Health Research Involving Human Subjects of the Ministry of Health, Labor and Welfare of Japan and the Declaration of Helsinki.

Consent
Written informed consent was obtained from the patient's parents.

Conflicts of Interest
e authors have no con icts of interest to declare with regard to this work.