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Myelodysplastic syndrome

An MDS-derived cell line and a series of its sublines serve as an in vitro model for the leukemic evolution of MDS

Leukemia volume 32, pages 1846–1850 (2018)Cite this article

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We performed comprehensive, comparative exome analyses on the series of samples by the next-generation sequencing (NGS) method. Total effective reads per sample ranged from 35,548,000 to 58,711,000 and the average sequencing depth ranged from 46 to 77. Candidate somatic mutations, excluding dbSNP, were detected in 406 genes, and 178 mutations were found in common in all ten samples. Main results are summarized in Supplementary Table 3. As notable mutations, a homozygous, splice donor site mutation of TP53(c.672+1G>A; COSM6906 in COSMIC) was present in all samples, including Pt-BM, together with homozygous TP53(c.74+14T>C) mutation. MDS-L lost normal TP53 protein probably due to these mutations (Supplementary Figure 2). CEBPA(Q311stop) mutation (heterozygous; COSM29221) and NRAS(G12A) mutation (heterozygous; COSM565) were not detected in Pt-BM, but they emerged clonally at MDS-Gen stage, and both mutations were inherited by all subsequent cell lines.

Whether CEBPA–mutant and NRAS–mutant clones were originally latent in Pt-BM or whether these mutations newly emerged during in vitro culture is an important question. To find the answer, we performed ultra-deep-targeted sequencing of CEBPA and NRAS in Pt-BM, and the results were as follows: (1) as for CEBPA, a single-allele 931C>T(Q311stop) mutation was detected in 181,059 counts out of 4,044,666 coverages. The detection frequency was therefore ~4.5%, and considering that it was a heterozygous mutation, the mutant clone must have accounted for ~9% of Pt-BM; (2) as for NRAS, no 35G>C(G12A) mutation count was detected at all out of 4,712,760 coverages. It implied that NRAS–mutant clone was completely absent from Pt-BM. It was therefore demonstrated that CEBPA mutation was originally present in a part of TP53-mutated bone marrow fractions and this clone was selected during IL-3-containing culture, and it was further suggested that NRAS mutation emerged by chance on the CEBPA–mutant clone during in vitro culture. Such accumulated mutations could generate MDS cell lines.

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Acknowledgements

The authors thank Dr. Takanori Ueda (Fukui Medical University, Fukui, Japan) for his outstanding supervision and contribution to ethical approval, Drs. Hiroya Kirimura and Kengo Gotoh (Central Research Laboratories, Sysmex Corporation, Hyogo, Japan) for genetic analysis. The authors also thank Ms. Aki Kuyama for editorial assistance. This work was supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI) and Kawasaki Medical School Project Grant.

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Authors and Affiliations

  1. Department of Laboratory Medicine, Kawasaki Medical School, Okayama, Japan

    Jun-ichiro Kida, Takayuki Tsujioka, Shin-ichiro Suemori, Shuichiro Okamoto, Kanae Sakakibara, Akira Kitanaka & Kaoru Tohyama

  2. Central Research Laboratories, Sysmex Corporation, Hyogo, Japan

    Takayuki Takahata

  3. Department of Hematology and Oncology, Faculty of Medical Sciences, University of Fukui, Fukui, Japan

    Takahiro Yamauchi

  4. Division of Biochemistry, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Hyogo, Japan

    Yumi Tohyama

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  1. Jun-ichiro Kida
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Correspondence to Kaoru Tohyama.

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Kida, Ji., Tsujioka, T., Suemori, Si. et al. An MDS-derived cell line and a series of its sublines serve as an in vitro model for the leukemic evolution of MDS. Leukemia 32, 1846–1850 (2018). https://doi.org/10.1038/s41375-018-0189-7

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