Press Release2023 年1 月 19 日

新聞社
支社 御中
上武大学副学長

林 泰秀

小児急性骨髄性白血病の新たな予後因子の同定
概要

上武大学医学生理学研究所の林泰秀副所長(上武大学副学長)，群馬県立小児医療センター血液腫瘍科の鏑木多映子部長，横浜市立大学附属病院小児科の柴徳生准教授らは，日本小児がん研究グループ(JCCG)が実施する急性骨髄性白血病（Acute myeloid leukemia; AML）の臨床試験に登録され治療を受けた小児患者に対し、次世代シーケンサーを用いた遺伝子解析を行い，小児 AML
において Upstream binding transcription factor (UBTF)遺伝子の 遺伝子内縦列重複変異(Internal tandem duplication, ITD): UBTF-ITD が新たな予後不良因子である可能性を報告しました．
AML は血液中に存在する白血球ががん化する血液がんの一種です．小児 AML ではこれまでの研究で RUNX1::RUNX1T1 融合遺伝子や、FLT3-ITD 遺伝子変異などの予後予測因子*1 が同定されており、疾患リスクに合わせた治療を行う層別化治療*2 が進められてきました。しかしながら、既存の予後予測因子を同定できない症例も多く存在し、治療成績の向上のためには新規の予後予測因子の同定を行うことが重要とされています。
今回の遺伝子解析の結果、小児初発 AML の 503 例中 6 例に UBTF-ITD を同定しました。

UBTF-ITD を有する症例は小児 AML でしばしば認められる 8 番染色体が 1 本多い 8 トリソミーとの合併が多く、全生存率*3、無イベント生存率*4 ともに有意に不良であることがわかりました。
これまで、8 トリソミー陽性例には予後良好例、不良例が混在していましたが、今回の UBTF-ITDの同定により 8 トリソミー陽性例の予後を正確に予測することが可能となりました。これらの結果は昨年米国の小児再発 AML を中心とした解析からも同様の報告がなされており、小児 AMLにおける UBTF-ITD が重要な予後因子であることを示唆しています。今後の症例数の蓄積により予後因子として確立することで正確な層別化治療や新規治療薬の開発につながり小児 AML の治療成績の向上に結び付くことが期待されます。
本研究成果は、科学誌『Genes chromosomes and Cancer』に掲載されます。
オンライン版：https://onlinelibrary.wiley.com/doi/10.1002/gcc.23110

研究に関するお問い合わせ：上武大学医学生理学研究所 副所長(上武大学副学長)

取材に関するお問い合わせ：林 泰秀

学校法人学文館上武大学

TEL: 0270-32-1011, FAX: 0270-32-1126, E-mail: hayashiy@jobu.ac.jp,

法人本部または群馬県立小児医療センター 血液腫瘍科部長 鏑木

TEL：0274-42-1183

多映子

TEL: 0279-52-3551, FAX: 0279-52-7333, E-mail: taeko0715@gmail.com〒370-1393 群馬県高崎市新町 270-1
FAX：0274-42-5202

Jobu University

Press Release【ポイント】
 小児 AML503 例中 6 例(1.2%)に UBTF-ITD を同定し、成人 AML には認められず小児 AML に特徴的な遺伝子異常と考えられました。
 UBTF-ITD を有する AML の特徴として、FLT3-ITD、PRDM16 遺伝子高発現※5、WT1 遺伝子変異、8トリソミーとの合併が多くみられることがわかりました。
 本研究の結果は 2022 年度に米国から報告された小児再発 AML で同定された UBTF-ITD 症例と同様の分子生物学的特徴や予後の傾向を示しており、UBTF-ITD が小児 AML において重要な予後不良因子である可能性が示されました。
 UBTF-ITD が予後因子として確立することで適切な層別化や新規治療薬の開発を目指すことが期待されます。
【研究の背景】
AML は血液のがんである白血病の一種で、小児白血病の中では急性リンパ性白血病の次に多い疾患です。AML においてはこれまで数多くの遺伝子解析が行われ、予後良好因子である RUNX1::RUNX1T1、
CBFB::MYH11 といった融合遺伝子や予後不良因子である FLT3-ITD などの遺伝子変異が複数同定されています。小児 AML ではそれらの予後因子や治療反応性を組み合わせた層別化治療が行われており、長期生存率は 60-70％まで上昇していますが、30-40%の患者さんは再発や死亡に至り、急性リンパ性白血病の生存率が 80-90%であることと比べると未だに十分な治療成績とは言えない疾患です。近年の遺伝子解析研究により多数の遺伝子変異や融合遺伝子が予後因子として同定され、以前と比較しリスクの層別化も進んでいますが、いまだに明らかな予後因子を持たずに適切な層別化治療が受けられない症例が存在します。新規の予後因子を同定して、より適切な層別化治療を行うことが小児 AML の治療成績向上のために重要とされています。
【研究の方法と結果】
本研究グループは、小児 AML131 例に対して次世代シーケンサーを用いて遺伝子解析を行ったところ、
UBTF-ITD という新しい遺伝子異常を同定しました。多くの症例で解析するため、小児 AML503 例、成人 AML175 例、小児悪性腫瘍(AML、急性リンパ性白血病、神経芽腫)の細胞株 65 株を対象にサンガー
シーケンス法という方法で解析したところ、小児 AML503 例中 6 例(1.2%)に UBTF-ITD を認めましたが、成人 AML には一例も認めませんでした。UBTF-ITD を有する AML の症例を詳細に解析したところ、
FLT3-ITD、WT1 遺伝子変異、PRDM16 遺伝子高発現、8 トリソミーといった遺伝子異常、染色体異常との合併が多いことがわかりました(図 1)。次に小児 AML139 例を対象に RNA シークエンスによる遺伝
子発現解析を行ったところ、UBTF-ITD を有する症例は、近年予後不良との関連が注目されている
PRDM16 遺伝子の発現が高く、他の PRDM16 高発現の症例と比較しても UBTF-ITD を有する症例はPRDM16 遺伝子の発現程度がより高いことがわかりました。
UBTF-ITD を有する症例とそれ以外の症例の予後を比較すると、UBTF-ITD を有する症例は全生存率、無イベント生存率ともに不良であることがわかりました(図 2)。また、8 トリソミーの症例の中で比較すると、通常予後因子として扱われない 8 トリソミーですが、UBTF-ITD を有する 8 トリソミーの症例は有意に予後不良であることがわかりました。8 トリソミーには他の染色体異常を合併することも知られていますが、UBTF-ITD を有する症例には他の染色体異常がなく、特徴的な集団であると示唆されました。UBTF-ITD は昨年米国の再発 AML を中心とした解析で初めて詳細に報告され、併存する分子生物学的異常や遺伝子発現パターン、予後への影響などが我々の結果と同様であり、今回の解析で再現性を確認することができました。UBTF は ITD 以外に急性リンパ性白血病では他の遺伝子と融合するキメラ

遺伝子を作ることが最近報告されて注目されており、血液腫瘍において重要な遺伝子である可能性が示
唆されています。UBTF-ITD は小児 AML おいて重要な予後因子となる可能性があります。

図１A UBTF-ITD を有する症例の分子生物学的特徴と臨床的特徴
UBTF-ITD を有する 6 症例を 6 列に示している。8 トリソミー、FLT3-ITD、WT1 遺伝子変異、PRDM16 遺伝子高発現との合併を多く認めた。
図１B 各症例の UBTF-ITD の遺伝子配列
各症例で同定された UBTF-ITD の遺伝子配列を示している。各症例の上段は塩基配列、下段はアミノ酸配列を示している。
いずれも Exon 13 内に重複を認め(黄色部分)、全症例に共通した重複配列(点線四角)を有していた。

図２．UBTF-ITD の有無による生存率の比較
UBTF-ITD を有する症例は全生存率(Overall survival)、無イベント生存率(Event-free survival)とも有意に不良であった。

【研究成果の意義と今後の展望】
本研究の成果として、UBTF-ITD が小児 AML における予後因子として確立した場合、より適切な層別化治療に結び付く可能性や、治療標的としての薬剤開発につながる可能性があり、今後の小児 AML の治療成績の向上が期待されます。

【論文情報】
掲載誌：Genes Chromosomes Cancer. 2022 Nov 30. doi: 10.1002/gcc.23110. Online ahead of print.
論文タイトル：UBTF-internal tandem duplication as a novel poor prognostic factor in pediatric acute myeloid
leukemia
著者：Kaburagi T, Shiba N, Yamato G, Yoshida K, Tabuchi K, Ohki K, Ishikita E, Hara Y, Shiraishi Y, Kawasaki
H, Sotomatsu M, Takizawa T, Taki T, Kiyokawa N, Tomizawa D, Horibe K, Miyano S, Taga T, Adachi S, Ogawa
S, Hayashi Y.
（和訳）
掲載誌：Genes Chromosomes Cancer 誌 2022 年 11 月オンライン doi: 10.1002/gcc.23110.、発行は 2023 年
1 月予定
論文タイトル：UBTF-ITD は小児急性骨髄性白血病における予後不良因子である
著者：鏑木 多映子、柴 徳生、大和 玄季、吉田 健一、田渕
白石 友一、河崎 裕英、外松 学、滝沢健、大木 健太郎、石北 悦子、原 勇介、琢己、滝 智彦、清河 信敬、富澤 大輔、堀部 敬三、宮野 悟、多賀 崇、足立 壮一、小川 誠司、林 泰秀【用語解説】※１

予後因子：治療後、その病気の状態がどうなるかを判断するための因子で、遺伝子異常の有無などが含まれる。
※２

層別化治療：予後因子に基づいて患者を分類し、それぞれのリスクに応じた最適と思われる治療を行うこと。
※３

全生存率：診断されてから一定の期間が経過した後に生存している人の割合。

※４

無イベント生存率：診断されてから一定の期間が経過した後、現病の進行や再発なく生存している人の割合。
※５

遺伝子高発現：細胞の中で DNA の遺伝情報をもとにたんぱく質が合成される際、通常よりもその合成が増強していること。

学校法人

学文館

上武大学

ウェブサイト

http://www.jobu.ac.jp/index.html○法人本部 〒370-1393 群馬県高崎市新町 270-1

TEL：0274-42-1183

FAX：0274-42-5202

Revised: 25 November 2022

Accepted: 25 November 2022

DOI: 10.1002/gcc.23110

RESEARCH ARTICLE

UBTF-internal tandem duplication as a novel poor prognostic
factor in pediatric acute myeloid leukemia
Taeko Kaburagi 1,2|

Norio Shiba 3 |Genki Yamato 1,2|

Kenichi Yoshida 4 || Etsuko Ishikita 1,2 | Yusuke Hara 2
Ken Tabuchi 5 | Kentaro Ohki 6
Yuichi Shiraishi 7 | Hirohide Kawasaki 1 | Manabu Sotomatsu 1 |Takumi Takizawa 2 |Daisuke Tomizawa 9
Souichi Adachi 13

Tomohiko Taki 8 | Nobutaka Kiyokawa 6|1011| Keizo Horibe| Satoru Miyano| Takashi Taga 12 || Seishi Ogawa 4,14,15 |Yasuhide Hayashi 1,16

1Department of Hematology/Oncology, Gunma Children's Medical Center, Gunma, Japan2

Department of Pediatrics, Gunma University Graduate School of Medicine, Gunma, Japan3

Department of Pediatrics, Yokohama City University Graduate School of Medicine, Kanagawa, Japan

4

Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

5

Department of Pediatrics, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan

6

Department of Pediatric Hematology and Oncology Research, National Research institute for Child Health and Development, Tokyo, Japan

7

Division of Genome Analysis Platform Development, National Cancer Center Research Institute, Tokyo, Japan

8

Department of Medical Technology, Kyorin University Faculty of Health Sciences, Tokyo, Japan

9

Division of Leukemia and Lymphoma, Children's Cancer Center, National Center for Child Health and Development, Tokyo, Japan

10

Clinical Research Center, National Hospital Organization Nagoya Medical Center, Aichi, Japan

11

M&D Data Science Center, Tokyo Medical and Dental University, Tokyo, Japan

12

Department of Pediatrics, Shiga University of Medical Science, Shiga, Japan

13

Human Health Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan

14

Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institute, Stockholm, Sweden

15

Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan

16

Institute of Physiology and Medicine, Jobu University, Gunma, Japan

Correspondence
Yasuhide Hayashi, Institute of Physiology andMedicine, Jobu University, 270-1 Shinmachi,
Takasaki, 370-1393 Gunma, Japan.
Email: hayashiy-tky@umin.ac.jp; hayashiy@jobu.ac.jp
Funding information
the Japan Society for the Promotion of Science(KAKENHI grants 17K10130, 18H06234,
19K21333, 20K08744, 20K17414); a grant for
project for development of innovative researchon cancer therapeutics (P-DIRECT) from theJapan Agency for Medical Research andDevelopment (AMED; JP16ck0106064,
JP19ck0106329, JP21ck0106634h); a Grantin-Aid for Scientific Research on Innovative
Areas from the Ministry of Health, Labor andWelfare of Japan (15H05909); a Grants forClinical Cancer Research from the Ministry ofGenes Chromosomes Cancer. 2022;1–8.

|

Abstract
The prognosis of pediatric acute myeloid leukemia (AML) has improved via stratification therapy. However, relapse or death occurs in 30%–40% of cases. Novel genetic
factors for pediatric AML need to be elucidated to improve prognosis. We detectedrecurrent internal tandem duplication in upstream binding transcription factor (UBTFITD) in 1.2% (6/503) of Japanese pediatric patients with de novo AML. No UBTF-ITD
was detected in 175 adult patients with AML or in 65 cell lines that included 15 AML,
39 acute lymphoblastic leukemia, five chronic myeloid leukemia, and six neuroblastoma
cell lines. All UBTF-ITDs were found in exon 13 and shared a duplicated region. UBTFITD was more frequently detected in patients with trisomy 8, FLT3-ITD, WT1 mutation,
and/or high PRDM16 expression (trisomy 8, 3/6; FLT3-ITD, 5/6; WT1 mutation, 2/6;
and high PRDM16 expression, 6/6). Gene expression patterns of patients with UBTFITD were similar to those of patients with NUP98::NSD1 or FUS::ERG. Survival analysis
wileyonlinelibrary.com/journal/gcc© 2022 Wiley Periodicals LLC.

110982264, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/gcc.23110 by Cochrane Japan, Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Received: 10 June 2022

KABURAGI ET AL.

Health, Labour and Welfare of Japan:
H17-GanRinsho-004, H20-GanRinsho-Ippan017, H23-GanRinsho-Ippan-014; a research
grant from the Japanese Society of
Hematology; the Kawano Masanori Memorial
Public Interest Incorporated Foundation for
Promotion of Pediatricsof the AML-05 cohort revealed that patients with UBTF-ITD had worse outcomes thanthose without UBTF-ITD (3-year event-free survival, 20% vs. 55%; 3-year overall survival, 40% vs. 74%). Moreover, among the 27 patients with trisomy 8, all three patientswith UBTF -ITD had a poor prognosis resulting in early events (relapse or non-complete
remission) within 1 year. Our findings suggest that UBTF-ITD may be a novel and significant prognostic factor for pediatric patients with AML.
KEYWORDS

AML, FLT3-ITD, pediatric, PRDM16, trisomy 8, UBTF-ITD

1

I N T RO DU CT I O N|

total of 280 in the AML-99 trial and 369 from a total of 443 patientsin the AML-05 trial. The clinical characteristics of the patients and a

The prognosis of pediatric acute myeloid leukemia (AML) has

comparison between available and unavailable samples in each study

improved owing to stratification therapy using cytogenetic aberra-

are described in Tables S1–S3. Patients diagnosed with acute promye-tions and therapeutic response.1 However, there are still poor progno-

locytic leukemia, Down syndrome-associated AML, and secondary

sis cases for which the detailed molecular background remains

AML were excluded from these studies. Details pertaining to the diag-

unknown. Thus, further investigation is needed to identify novel prog-

nosis, treatment, and risk stratification have been previously

nostic factors in pediatric AML.

reported.6,7 The treatment protocols and procedures for data andWhile FLT3-ITD has been identified as an important prognostic

sample collection were approved by the institutional review boards offactor in pediatric and adult AML, evidence has also shown that FLT3-

each participating institution; written informed consent was obtained

ITD alone does not determine the prognosis, which has been found tofrom all patients or their parents/guardians. These studies were con-depend on other coexisting genetic factors.2–4 Our previous RNA

ducted in accordance with the Declaration of Helsinki and approved

sequencing analysis of 139 pediatric AML found that 37 of 47 patientsby the institutional review board of the Gunma Children's Medicalwith FLT3-ITD had some type of fusion gene, but no other factors

Center and the ethical review board of the JPLSG. Furthermore,

5

were identified in the remaining 10 patients. Focusing on these

175 adult patients with AML were enrolled as a control cohort.

10 patients with FLT3-ITD without any fusion genes, we performed acustom panel sequencing on 131 pediatric AML and identified recurrent internal tandem duplication in upstream binding transcription fac-

2.2|

Cell lines

tor (UBTF-ITD) in 3 of 10 patients with FLT3-ITD without any fusion
genes. UBTF encodes a member of the HMG-box DNA-binding pro-

The following 65 cell lines were analyzed: 15 AML, 39 acute lympho-

tein family and plays a critical role in ribosomal RNA transcription. We

blastic leukemia, five chronic myeloid leukemia, and six neuroblastoma

further analyzed UBTF-ITD in an expanded cohort comprising pediat-cell lines. Detailed information regarding the cell lines is presented in

ric AML, adult AML, and leukemia and neuroblastoma cell lines by

Table S4.

Sanger sequencing and investigated the correlation between UBTFITD and other cytogenetic aberrations, gene expressions, clinical features, and prognosis.

2.3|

Custom panel sequencing

Custom panel sequencing was performed in 131 of 369 patients in

2

MATERIALS AND METHODS|

the AML-05 study. This panel contained 343 genes that have beenpreviously reported as driver or targetable genes in hematological

2.1|

Patients

malignancies or solid tumors, including UBTF, which was reported in
integrated analysis in erythroleukemia.8 All coding exons were cap-

This study enrolled patients with de novo AML who participated in

tured using the SureSelect custom kit (Agilent Technologies, Santathe Japanese AML-99 trial conducted by the Japanese Childhood

Clara, CA) and sequenced using the Hiseq 2500.

AML Cooperative Study from January 2000 to December 2002, as
well as the AML-05 trial conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) from November 2006 to

2.4|

Sanger sequencing for UBTF-ITD

December 2010.6,7 The AML-05 trial is registered with the UMIN
Clinical Trials Registry (UMIN-CTR, http://www.umin.ac.jp/ctr/index.

Analysis of UBTF-ITD was performed via polymerase chain reaction

htm, number UMIN000000511). A total of 503 patients with leukemic(PCR) followed by Sanger sequencing. Sanger sequencing was per-samples were included in this study. There were 134 patients from aformed to detect UBTF-ITD in 369 patients from the AML-05 study,

10982264, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/gcc.23110 by Cochrane Japan, Wiley Online Library on [07/12/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

2

+

+

+



+

and 67 cell lines. The following primers were used: forward primer,



Dead

157 patients from the AML-99 study, 175 adult patients with AML,

47,XX,+8[20]47,XY,+8[20]

46,XX
M0

methods are described in Supplementary Methods. Inter-group differ-

M5a

FAB

cal University, Saitama, Japan) was used for all analyze.10 Detailed

47,XX,+8[20]The EZR software (version 1.35; Saitama Medical Center, Jichi Medi-

M1

Statistical methods

M1|

47,XY,+8[1]/46,XY[19]2.7

Karyotypevious report.5

M4

characteristics and methods are presented in Table S6 and in our pre-

M6a

Genome-Phenome Archive (EGAS00001003701). Detailed patient

46,XX[20]AML-05 study. The RNA-seq data are available at the European

25.7

95.9

Event-free survival (EFS) was defined as the time from diagnosis to168.1

defined as the time from diagnosis to death or the last follow-up.

87.5

were used to estimate the survival rate. Overall survival (OS) was

7.0

Mann–Whitney U tests. The Kaplan–Meier method and log-rank test

1.1

WBC, 103/μl

ences in clinical characteristics were analyzed using Fisher's exact and175 adult AMLs and 65 cell lines with leukemia and neuroblastoma. All

8.5

8
F

F
36 bp
27 bp

39 bp

AML-05-365

AML-99-154

15 bp

7.4

8.3

M

F

M

3 bp

12.8

14.9
11 bp

F



90 bp

503 (1.2%) pediatric AML cases were found to have UBTF-ITD (Table 1
and Figure 1A). In contrast, no UBTF-ITDs were detected in the

TABLE 1

AML-99 study via Sanger sequencing, respectively. Thus, six of81 bp

ITDs in two patients from the AML-05 study and one patient from the

AML-05-67

custom panel sequencing. Thereafter, we detected additional UBTF-

AML-05-275

Three patients with UBTF-ITD were detected from 131 patients via

10 bp

Detection of UBTF-ITD in pediatric AML

38 bp

|

AML-05-47

3.1

RESULTS

|

40 bp

3

AML-05-21<0.05 were considered indicative of statistical significance.

Sexvarious genetic abnormalities. For all analyses, two tailed p values ofInsertion

Wallis test was used to compare gene expression levels among the

Duplicated lengthinterval were estimated using Cox regression analysis. The Kruskal–

Clinical characteristics of patients with UBTF-ITD

last contact for all other patients. Hazard ratio and 95% confidence

UPN

death) for patients who experienced treatment failure or the date ofAge, year

the date of failure (induction failure, relapse, second malignancy, orAbbreviations: CR, complete remission; FAB, French–American–British classification; N/A, not available; SCT, stem cell transplantation; WBC, white blood cell counts; UPN, unique patient number.

+

+
+

+

+
+

+

+
+

+



+


+



+

+

+
+
+

+

+

RNA sequencing was performed in 139 of the 369 patients from the+

RNA sequencing

+

|

+

2.6

+

Detailed methods have been reported previously.9

were analyzed using Sanger sequencing and quantitative RT-PCR.

High PRDM16 expression

Various gene mutations, gene rearrangements, and gene expressions

FLT3-ITD

CR

SCT

2.5 | Molecular characterization other than
UBTF-ITD+

RelapseCTTCCCGCCTTC-30 .



50 -ACCCTCTGCACCTGGACA-30 and reverse primer, 50 -TGACAG

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KABURAGI ET AL.

KABURAGI ET AL.

F I G U R E 1 (A) Molecular and cytogenetic aberrations in six pediatric acute myeloid leukemia (AML) with UBTF-ITD in the AML-05 and AML99 studies. Among the six patients with UBTF-ITD, four had trisomy 8, and the other two had a normal karyotype. Moreover, five and two of thesix patients with UBTF-ITD had concomitant FLT3-ITD or WT1 mutations, respectively. High PRDM16 expression was also detected in all patientswith UBTF-ITD. Each column displays the cytogenetic aberration pattern and clinical status of an individual sample. Light blue indicates UBTF-ITD.
Green indicates chromosomal aberrations. Yellow indicates genetic aberrations. Blue indicates gene expression. Gray indicates a clinical outcome.
Blanks indicate the absence of a chromosomal aberration, genetic alteration, or prognostic event. Light and dark purple indicate risk classification.
(B) UBTF-ITD sequences in each patient. The image shows the sequences of UBTF-ITD in exon 13. The top row of each case shows thenucleotide sequences, whereas the bottom row shows the amino acid sequence. Duplicated regions are depicted in red characters and a yellow
marker. Commonly duplicated regions are indicated by the dotted line.

UBTF-ITDs were detected in exon 13 and had common duplicated

3, including CTNNB1 and BCL3. Thereafter, we compared generegion (Figure 1B).

expressions between AML with and without UBTF-ITD. The expression of UBTF, PRDM16, HOXB5, HIST1H1C, and CLU differed significantly (Figure S1). Finally, gene expressions were compared among

3.2 | Clinical and cytogenetic characteristics ofpatients with UBTF-ITD

representative genetic abnormalities, including NUP98::NSD1, FUS::
ERG, NPM1, biallelic CEBPA mutations, KMT2A rearrangements, orothers (Figure S2). UBTF expression tended to differ among these

The landscape of patients with UBTF-ITD is described in Figure 1A.

seven groups (p = 0.057, Figure S2), and the expression of PRDM16,

Trisomy 8, FLT3-ITD, WT1 mutation, and high PRDM16 expression

HOXA9, HOXB5, FLT3, CEBPA, HIST1H1C, and CLU differed signifi-

were more frequently detected in patients with UBTF-ITD from thecantly among these groups. The expression levels of these genes in

AML-05 study, respectively (trisomy 8, n = 3, p = 0.003; FLT3-ITD,

cases with UBTF-ITD were similar to those in AML with NUP98::NSD1

n = 4, p = 0.001; WT1 mutation, n = 2, p = 0.039; high PRDM16

or NPM1 mutation (Figure S2). Among them, the expression level ofexpression, n = 5, p = 0.001) (Table 2).

CLU was higher in cases with UBTF-ITD or NUP98::NSD1 but lower inpatients with NPM1 mutation. Intriguingly, patients with UBTF-ITD
had a significantly higher PRDM16 expression level than those without

3.3 | Gene expression analysis of cases with
UBTF-ITDUBTF-ITD among the 64 patients with high PRDM16 expression(p = 0.010, Figure S3).

Unsupervised hierarchical clustering analysis of the 139 pediatric
AML is shown in Figure 2A,B. Five clusters were generated, with eachcluster having unique characteristics related to gene alterations. Cases

3.4 | Survival analysis of patients with UBTF-ITDpositive AML in the AML-05 study

with UBTF-ITD were categorized into clusters 1 and 3 (Figure 2A).
Region B consisted of HISH1Hs and HOXAs, the gene expression ofSurvival analysis was performed in the AML-05 study using thewhich were higher in clusters 1A and 3 (Figure 2B). In contrast, genes

Kaplan–Meier method to reveal the prognostic impact of UBTF-ITD.

belonging to region A were lower in clusters 1A and 3. Genes catego-

Notably, patients with UBTF-ITD had significantly poorer outcomes inrized to region C differed significantly between clusters 1A andterms of both EFS and OS than those without UBTF-ITD (3-year EFS,

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4TABLE 2
UBTF-ITD

Clinical characteristics of patients with and without

4|

DI SCU SSIONUBTF-ITD(n = 5)w/o UBTF-ITD(n = 364)

UBTF-ITD was detected in 6 (1.2%) out of 503 pediatric AML cases

Age median at diagnosis,
year (range)

8.5 (7.4–14.9)

7.8 (0.0–17.9)

relapsed pediatric patients with AML.11,12 As indicated in previous

Sex, Male, n (%)

2 (40)

192 (53)

in exon 13, resulting in in-frame changes.11,12 Consistent with previ-

WBC median, 10 /L(range)87.5 (1.1–168.1)

20.1 (0.62–985.0)

ous reports,11,12 UBTF-ITD was detected specifically in pediatricStem celltransplantation, n4171

was strongly associated with trisomy 8, FLT-ITD, WT1 mutation, andRelapse, n3

129

ITD also had a significantly worse prognosis, which is consistent with9in this study. UBTF-ITD has recently been reported mainly instudies, all these ITDs coexisted with various insertions and deletions

AML in this study. Also similar to the previous report,11 UBTF-ITD
high PRDM16 expression (Table 2) in this study. Patients with UBTF-

FAB classification, n

the previous report.11 Patients with FLT3-ITD were classified as

M0

0

7

high-risk and were all indicated for stem cell transplantation in this

M1

2

46

study.6 High PRDM16 expression was reported to be associated with

M2

0

105

poor prognosis in our previous study.13 Furthermore, we also

M4

1

55

showed, that among patients with pediatric AML, the prognosis of

M5

1

72

those with FLT3-ITD can be divided according to the presence or

M6

1

7

M7

0

33

absence of high PRDM16 expression.14 Thus, high PRDM16 expression has recently been recognized as a strong poor prognostic factor.
Considering that patients with UBTF-ITD concomitantly had these

Risk classification, npoor prognostic factors, it was difficult to determine the significance

Low

0

126

Intermediate

0

144

High

3

46

prognosis given that patients with UBTF-ITD had more higher

Non-complete
remission

248

expression of PRDM16 (Figure S3). Further analysis of UBTF-ITD

of UBTF-ITD alone. However, our findings suggested that UBTF-ITD
enhanced the expression of PRDM16 and contributed to the poor

should better elucidate the mechanism by which PRDM16 expression

Cytogenetic features, n

is enhanced in leukemic cells. Furthermore, gene expression analysis

Normal karyotype

1

69

revealed that patients with UBTF-ITD expression patterns were simi-

Trisomy 8

324

lar to those with NUP98::NSD1 or FUS::ERG (Figure 2A). UBTF-ITD

RUNX1-RUNX1T1

0106

can be considered a novel abnormality characteristic to a similar

CBFB-MYH11031

group with a poor prognosis, such as those with NUP98::NSD1 or

KMT2Arearrangement

055

FUS::ERG. In particular, the prognosis of patients with trisomy 8 can

WT1 mutation

2

23

UBTF-ITD, NUP98::NSD1, or FUS::ERG (Figure 4C). Trisomy 8 is

FLT3-ITD

4

43

sometimes detected with other cytogenetic aberrations such as

High PRDM16
expression

579

be clearly determined according to the presence or absence ofAbbreviations: FAB, French–American–British; WBC, white blood cellcounts.

KMT2A rearrangement or CBFB::MYH11. In fact, 17 of 27 patientswith trisomy 8 harbored additional cytogenetic abnormalities. Moreover, the patients with UBTF-ITD had no other cytogenetic aberrations other than trisomy 8. In a previous report, despite the highfrequency of FLT3-ITD in patients with trisomy 8 alone, no signifi-

20% vs. 55%; 3-year OS, 40% vs. 74%) (Figure 3). Further analyses

cant difference was detected between the prognosis of patients with

were performed, focusing on patients with trisomy 8 (n = 27). The

trisomy 8 alone and those with trisomy 8 harboring other cytoge-clinical characteristics of patients with trisomy 8 are described in

netic alterations.15 On the contrary, the results of this study showed,

Figure 4A and Table S7. All three patients with trisomy 8 had no otherthat among the patients with trisomy 8 alone, those with UBTF-ITD

cytogenetic aberrations, such as KMT2A rearrangement or CBFB::

may have a worse prognosis.

MYH11 (Figure 4A). Furthermore, all three patients with trisomy 8 with

Given the small number of patients, it is difficult to determine the

UBTF-ITD had significantly worse outcomes, resulting in early events

clinical significance of UBTF -ITD based on the results of this study(relapse or non-complete remission) within 1 year (Figure 4B). Patientsalone. However, the results of this study are consistent with previous

with trisomy 8 with NUP98::NSD1, FUS::ERG, or UBTF-ITD had an

studies.11 Although further cases are needed to establish the clinicalextremely poor prognosis, whereas patients with trisomy 8 without

significance of UBTF-ITD, it may be useful for more accurate stratifica-

those had a favorable prognosis (3-year EFS, 11% vs. 64%; 3-year OS,tion and may contribute toward improving the prognosis of pediatric

33% vs. 82%) (Figure 4C).

patients with AML.

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KABURAGI ET AL.

KABURAGI ET AL.

F I G U R E 2 (A) Unsupervised hierarchical clustering analysis of the 139 pediatric AML. The 139 cases were classified into five clusters. Eachcluster had the following characteristics: cluster 1A, AML with NPM1 mutation; cluster 1B, acute megakaryoblastic leukemia, and some cases withbiallelic CEBPA mutation, cluster 2, KMT2A rearrangement; cluster 3, NUP98::NSD1, UBTF-ITD, or RUNX1 mutation; cluster 4, some cases with
CEBPA biallelic mutation. (B) Distinct three regions from unsupervised hierarchical clustering analysis of the 139 pediatric AML. Region A
consisted of low-expression genes in clusters 1A and 3. Region B consisted of high-expression genes in clusters 1A and 3. Region C consisted ofgenes with significantly different expression patterns between clusters 1A and C.

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6

F I G U R E 3 Prognostic significance of
UBTF-ITD in pediatric AML from the
AML-05 study. The Kaplan–Meier curvesindicate the overall and event-freesurvival of patients with or without
UBTF-ITD.

F I G U R E 4 (A) Molecular and cytogenetic aberrations in 27 pediatric AMLs with trisomy 8 from the AML-05 study. Each column displays thecytogenetic aberration pattern and clinical status of an individual sample. Pink indicates trisomy 8. Light blue indicates UBTF-ITD. Green indicates
chromosomal aberrations. Yellow indicates genetic aberrations. Orange indicates gene expression. Gray indicates a clinical outcome. Blanks
indicate the absence of a chromosomal aberration, genetic alteration, or prognostic event. Light orange, pink, light purple, and dark purple indicate
risk classification. (B) Prognostic significance of UBTF-ITD in pediatric AML with trisomy 8 participated in the AML-05 study. Kaplan–Meiercurves indicate the overall and event-free survival of patients with or without UBTF-ITD in AML with trisomy 8. (C) Prognostic significance of
NUP98::NSD1, FUS::ERG, or UBTF-ITD among patients with AML with trisomy 8. Kaplan–Meier curves indicate the overall and event-free survivalof cases that are positive or negative for NUP98::NSD1, FUS::ERG, or UBTF-ITD.

AUTHOR CONTRIBUTIONS

Kiyokawa, Daisuke Tomizawa, Keizo Horibe, Takashi Taga, and Soui-

Taeko Kaburagi, Norio Shiba, Genki Yamato, Etsuko Ishikita, andchi Adachi provided patient samples and data. All authors critically

Yusuke Hara designed and performed the research, analyzed the data,

reviewed and revised the manuscript.

and wrote the article. Yasuhide Hayashi designed the research, led the
project, and wrote the article. Ken Tabuchi performed statistical anal-

ACKNOWLEDG MENTS

ysis. Kenichi Yoshida, Yuichi Shiraishi, Satoru Miyano, and Seishi

The authors thank Yuki Hoshino for her valuable assistance in per-Ogawa performed the research. Kentaro Ohki, Manabu Sotomatsu,

forming the experiments. The authors would like to thank Enago

Hirohide Kawasaki, Takumi Takizawa, Tomohiko Taki, Nobutaka(www.enago.jp) for the English language review. This work was

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7

KABURAGI ET AL.

KABURAGI ET AL.

supported by a Grant-in-Aid for Scientific Research on Innovative
Areas from the Ministry of Health, Labor and Welfare of Japan(15H05909), a grant for project for development of innovative
research on cancer therapeutics (P-DIRECT) from the Japan Agency
for Medical Research and Development (AMED; JP16ck0106064,
JP19ck0106329, JP21ck0106634h), the Japan Society for the Promotion of Science (KAKENHI grants 17K10130, 18H06234, 19K21333,
20K08744, 20K17414), a research grant from the Japanese Society of
Hematology, the Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics, and a Grants for ClinicalCancer Research from the Ministry of Health, Labour and Welfare ofJapan: H17-GanRinsho-004, H20-GanRinsho-Ippan-017, H23-GanRinsho-Ippan-014.
CONF LICT OF IN TE RE ST
The authors declare that they have no conflicts of interest.
DATA AVAI LAB ILITY S TATEMENT
The data that support the findings of this study are available from thecorresponding author upon reasonable request.
ORCID
Kentaro Ohki

https://orcid.org/0000-0003-2838-4555

Yusuke Hara

https://orcid.org/0000-0001-7271-3454

Nobutaka Kiyokawa

https://orcid.org/0000-0001-9310-2126

Daisuke Tomizawa

https://orcid.org/0000-0003-1520-7007

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SUPPORTING INF ORMATION
Additional supporting information can be found online in the Supporting Information section at the end of this article.

How to cite this article: Kaburagi T, Shiba N, Yamato G, et al.
UBTF-internal tandem duplication as a novel poor prognostic
factor in pediatric acute myeloid leukemia. Genes
Chromosomes Cancer. 2022;1‐8. doi:10.1002/gcc.23110

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