金沢大学がん 進展制御 研究所
腫瘍制御（旧：遺伝子診断）研究分野
研究のあゆみと業績 2022＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊
Annual Report 2022
Division of Translational and Clinical Oncology(previously: Division of Diagnostic Molecular Oncology)

Cancer Research Institute, Kanazawa University
Kanazawa, Japan

2022 年 12 月
December 2022

研究のあゆみと業績 2022

まえがき
本誌の編集は今回が 21 回目です（研究のあゆみと業績 2001 年と 2002 年は合冊のため）。
この間に研究分野開設 10 年（Decennial Report 2001-2010: Downstream, Present & Upstream）、
15 年（Quinquennial Report ）と 20 年（Vicennial Report 2016-2020）の記念誌を作りましたので、この手の編集は 24 回におよびます。外科と腫瘍学の教えを受けた磨伊正義先生（故人）が生前、心配なさっていたとはいえ、弱小体制ながらなんとか 22 年間は持ちこたえたようです。そして、23年に向かおうとしています。惰性で日記のようになってきても、もうしばらく編集を続けます。
今春、もう当たらないと思っていた研究費２件が採択され、ほっとしました（第８頁）。ところがこれと前後して３月 16 日に、病理学とその後の生き方を絶えず指導してくださった中西功夫先生（本学旧第一病理学：名誉教授）が他界されました。初期の本誌を届けに伺ったときには、「源さ．．．
んの文章は硬くてまわりくどくていかん。もう少し、柔らかく書けばいい。それでも、よくやっている」と云われました。今でも昨日のことのように思い出されます。この後は、研究室独自の成果が芳しくなく、淡々と日々を過ごしていましたところ、９月 19 日の朝、共同研究者の石渡俊行先生（東京都健康長寿医療センター研究所）から、松田陽子先生（香川大学腫瘍病理学：教授）の訃報を伝えるメールが届きました。2019年２月の教授内定のささやかなお祝いなどこれまでの交流を考えると、かけがえのない仲間を失ったとても
悲しい知らせでした。８月初旬に私が推薦した
第31回日本消化器癌発生学会：田原榮一賞に内定した直後だったのです。それでも、ご逝去後の11月に田原賞を受賞され、遺作となった共同研究論文が12月下旬にJ Natl Cancer Instに採択されたこと（第９頁：研究業績を参照）は、
私にとってはなによりの供養になったと思い、ご冥福を祈るしだいです。私と松田先生との交2019年2月27日 駅前居酒屋 狼煙（金沢駅前）
流の経緯については、研究のあゆみと業績 2021年にも掲載した当研究所ニュースレター（抜粋）を再掲します【附記２：第13-15頁】。
私自身は2021年末に、日本消化器内視鏡学会から第31回（2022年度）北陸支部セミナーを担当するように指名されました。そしてこの一年間、多くの旧知や新たにできた仲間の皆さんに助けられて、年明けの開催の目途がたちました。2008年には消化器・腫瘍外科から身を引いたものの、消化器内視鏡専門医、指導医としてなんとか持ちこたえている自身に課せられた任務と受けとめ、できるかぎりの役目をはたすよう努めます。
この年末が過ぎて来春には、大学での最後の年度を迎えます。年明けから１年３か月のあいだ、何をしようか思案することにします。それでは、これからもどうぞよろしくお願いします。
2022年１2月 年末〒920-0934 石川県金沢市宝町 13 番 1 号
進展
金沢大学がん 制御 研究所・腫瘍制御
金沢大学附属病院がんセンター（併任）金沢医科大学病院内視鏡科（非常勤）☎ 076-265-2792（直通），2798（事務，研究室）
📠📠📠📠 076-234-4529
📨📨📨📨 minamoto@staff.kanazawa-u.ac.jp

Division of Translational and Clinical Oncology
Cancer Research Institute and Cancer Center
Kanazawa University and Hospital
13-1 Takara-machi
Kanazawa 920-0934, Japan
Phone 81-76-265-2792 (office), 2798 (secretary)
Fax 81-76-234-4523
e-mail: minamoto@staff.kanazawa-u.ac.jp

HP（更新中）: http://www.kanazawa-u.ac.jp/~ganken/shuyoseigyo/index.html-1-1-

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野【目次】
研究スタッフ……………………………………………………………………… 3

共同研究者………………………………………………………………………… 42021 年のあゆみとできごと ……………………………………………………… 5
研究分野と活動の概要
研究費…………………………………………………………… 6……………………………………………………………………………… 8

研究業績 …………………………………………………………………………… 9
論文発表
学会発表
その他（地域・社会貢献など）
知的財産，関連活動など ………………………………………………………… 11
メディア・新聞報道など ………………………………………………………… 11【附記１】金沢大学公開講座：がん医療の最前線（抜粋）……………………… 1122
【附記２】金沢大がん研 News Letter（（抜粋）：共同研究者紹介 ………………… 13
修了
了 ……………………………………
…………………………………… 1166
【附記３】学位論文： 2022 年３月と 12 月 修-2-研究のあゆみと業績 2022

【研究メンバー】 （2022 年 12 月現在）
教授

源 利成

金沢大学附属病院がんセンター（併任）

助教

堂本貴寛

博士研究員

上原将大

たか ひろ

ま さ ひろデ ィ リ レ バ

ボ

リ

ド

ン

Diliraba Bolidong
さとし～2022 年 3 月

大学院

竹中 哲

大学院医学系研究科肝胆膵・移植外科学（博士課程）

太田亮介

大学院医薬保健学総合研究科 腫瘍制御学（～3 月）

まさのり

研究生

小竹優範

厚生連高岡病院 外科（～12 月）

研究支援員

浅香敦子

研究支援推進員

研究協力員

あさ い

旭井亮一

（株）凸版印刷研究所

川島篤弘

独立行政法人国立病院機構金沢医療センター 臨床検査科

ひろ の り藤沢弘範

独立行政法人国立病院機構金沢医療センター 脳神経外科

横井健二

米国 ヒューストンメソジスト研究所ナノ医療部門

しまさき

島崎猛夫
ひがし

東 朋美

かさしま

笠島里美
かつ よ し

宮下勝吉
なかじま

共同研究者

金沢医科大学総合医学研究所，消化器内科
金沢大学医薬保健研究域医学系 環境分子応答学（衛生学）
金沢大学医薬保健研究域保健学系 検査技術科学
福井県立病院 脳神経外科

中島日出夫

上尾中央総合病院 腫瘍内科

下崎真吾

医療法人社団 下崎整形外科医院

澤田 武

名古屋市立大学消化器・内分泌内科学

太田亮介

金沢大学大学院医薬保健学総合研究科 腫瘍制御（～3 月）

づか

塚 正彦

金沢大学医薬保健研究域医学系 法・社会医学（法医学）-3-3-

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野【共同研究者】 （2022 年現在で共同研究が稼動および予定しているもの．敬称略）
な か だ

教授

中田光俊

金沢大学医薬保健研究域医学系 脳機能制御学/脳神経外科学

教授

土屋弘行

金沢大学医薬保健研究域医学系 機能再建学／整形外科学

特任教授 山本憲男

金沢大学医薬保健研究域医学系 機能再建学／整形外科学

教授

竹村博文

金沢大学医薬保健研究域医学系 心血管外科学

教授

稲木紀幸

金沢大学医薬保健研究域医学系 消化管外科学

准教授

木下 淳

金沢大学医薬保健研究域医学系 消化管外科学

け い し

助教

中村慶史

金沢大学医薬保健研究域医学系 消化管外科学

教授

八木真太郎

金沢大学医薬保健研究域医学系 肝胆膵・移植外科学

講師

牧野 勇

金沢大学医薬保健研究域医学系 肝胆膵・移植外科学

教授

Richard Wong

金沢大学理工学研究域自然システム学系／ナノ生命科学研究所

准教授

宮下知治

金沢医科大学 一般・消化器外科学／腫瘍外科学

教授

曽我朋義

慶應義塾大学 先端生命科学研究所

教授

せん

竹田 扇

帝京大学医学部 解剖学

学部内准 吉村健太郎

山梨大学大学院総合研究部医学域 総合医科学センター 分子生

教授

物学研究室
いしわた

研究部長 石渡俊行

東京都健康長寿医療センター 老年病理学，高齢者がん研究教授

香川大学医学部病理病態・生体防御講座 腫瘍病理学（～9 月）

松田陽子（故人）
す い つ

ふとし

准教授

水津 太

香川大学医学部病理病態・生体防御講座 腫瘍病理学

教授

松下一之

千葉大学医学研究院 分子病態解析学，附属病院検査部

教授

や す し

佐々木泰史
え い じ

札幌医科大学 教養教育研究部門 医療人育成センター

准教授

久保田英嗣

名古屋市立大学消化器・内分泌内科学

助教

古田拓也

久留米大学医学部 病理学

研究員

小泉恵太

上尾中央総合病院

かみ

紙 健次郎

（株）ヒューマンメタボロームテクノロジーズ

教授

Ze’ev Ronai

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA

教授

Andy Giraud

豪州オーストラリア王立小児病院

Louise M. Judd

豪州オーストラリア王立小児病院

Trevelyan R. Menheniott 豪州オーストラリア王立小児病院
准教授

Phil Sutton

豪州オーストラリア王立小児病院

Serge Y. Fuchs

ペンシルヴェニア大学 生物学-4-

研究のあゆみと業績 2022

【2022 年のあゆみとできごと】
2022 年 3 月 31 日
【附記３】

・太田亮介君が大学院を修了し、医学博士の学位を取得。
課題：Integrated genetic and epigenetic analysis of cancer-related genes in
non-ampullary duodenal adenomas and intramucosal adenocarcinomas. J Pathol 252 (3): 330-42, 2020.

2022 年 3 月 31 日

・博士研究員：Dilireba Bolidong さんが退職。

2022 年 4 月 1 日

・科学研究費２件が採択された。
2022 年－2024 年度 基盤研究（Ｂ） 課題番号 22H03144
源 利成（代表），宮下知治（分担）課題：治療耐性膵がんの悪性形質を繋ぐ分子経路の解明と耐性制御法開発への応用
2022 年－2024 年度 基盤研究（C） 課題番号 22K07227
堂本貴寛（代表）
課題：抗がん剤耐性獲得膵がん細胞における悪性形質連関の解明と治療法開発

2022 年 6 月 11 日
【附記１】

・源 利成が金沢大学公開講座「がん医療の最前線」で講演した。

2022 年 9 月 4 日

・源 利成が第 38 回日本臨床細胞学会北陸連合会学術集会で講演した。

課題：大腸がんのしくみと医療を一緒に学びましょう
課題：膵がん難治性の細胞特性 ― その仕組みと私たちの取り組み ―2022 年 9 月 17 日
【附記２】

・共同研究者：松田陽子さん（香川大学医学部病理病態・生体防御講座
腫瘍病理学 教授）がご逝去された（享年 49 歳）。

2022 年 12 月 31 日 ・研究生：小竹優範君（厚生連高岡病院 外科）が研究期間を終え、論文【附記３】を提出して医学博士の学位を取得した。
課 題 ：LOH of the thymidylate synthase locus in combination withgenotype has prognostic and predictive significance in colorectal
cancer. Mol Clin Oncol 15: 235, 2021.

博士研究員：Dilireba Bolidong さん退職のときのささやかな食事会（2022 年 3 月 24 日：金沢まいもん寿司 白山インター店）-5-5-

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野【研究分野と活動の概要】
当研究分野は 1998 年に遺伝子診断の旧称で開設され、その後 24 年間にわたって、
消化器がんを中心にがんの多様な生物病態と腫瘍外科学的特性について、基礎と臨床を関係づけるかたちの研究を指向している。そして、その成果を難治がんや希少がんの病態解明と制御に応用するために、学内外のグループと共同研究を進める。

Division Summary
The mission of our division centers on laboratory and clinical research to develop thenovel strategies and modalities for diagnosis and treatment of the gastrointestinal (GI),refractory and rare cancer types including glioblastoma, bone and soft tissue sarcomas andpancreatic neuroendocrine neoplasms. Research projects are based on biological characteristics of individual tumor types that are relevant to their invasive and metastatic potential,
resistance to therapy, recurrence and outcome of patients. Our current efforts are focused on(1) research and development of the cancer therapy by targeting aberrant glycogen synthasekinase (GSK)3; (2) understanding of malignant phenotypes of cancer by investigating
pathological metabolic properties (eg., aerobic glycolysis, tumor-promoting autophagy); and(3) biological basis of GI and refractory cancer, all for clinical translation. We have been
also establishing the tissue material resources of human stomach and colorectal cancer forour own projects described above as well as for studies collaborating with our institutionaland many other research groups. During an immediate couple of years, we have obtained thepreliminary results indicating the putative roles of tumor GSK3 as a molecular node that isintersected by the pathways responsible for tumor invasion and resistance to therapy, thus
enforcing its potential as an emerging cancer therapeutic target. We are extending this project
toward investigation of the putative roles for GSK3 in promoting pancreatic neuroendocrine neoplasms as well as in interconnecting malignant phenotypes in pancreaticcancer with acquired chemoresistance. Following the project on GSK3, we have recently
identified a number of molecules potentially responsible for acquired resistance to gemcitabine in pancreatic cancer by comparing the cDNA microarray-based gene expression
between gemcitabine-sensitive BxPC-3 cells and BxPC-3-derived clones that acquired
stepwise resistance to gemcitabine. In addition to these projects, we have participated in thecollaborative studies showing the association of hTERT phosphorylation and longer
telomere length in cancer cells with worth prognosis in various cancer types.
＜2022 年の研究成果，進捗状況及び今後の計画＞
１．glycogen synthase kinase (GSK) 3β 阻害によるがん治療法の研究、開発
Wnt 経路抑制因子と認識されている GSK3 が固有の分子経路を介して、がんの悪性形質を-6-

研究のあゆみと業績 2022

推進することを系統的に示してきた。そして、GSK3 阻害のがん治療効果を細胞レベルと担がん動物で実証した。また、学内外の外科系グループと連携し、膵がん、膠芽腫や骨軟部肉腫などの難治，希少がんで高活性を示す GSK3 が，腫瘍浸潤性と治療（抗がん剤，放射線）不応性の悪性形質を連結することを見出した。一連の研究をもとに、GSK3 阻害薬品の転用と抗がん剤を併用するがん治療法を開発し、再発膠芽腫（附属病院脳神経外科）と進行膵がん（金沢医科大学
病院）を対象とする医師主導臨床研究によりその安全性と抗腫瘍効果を検証した。ついで、食道扁平上皮がんと抗がん剤耐性獲得膵がんに対する GSK3 阻害の治療効果とメカニズムを明らかにした。2022 年には GSK3 阻害によるがん治療の概念実証のため、膵内分泌腫瘍（PNET）の共同研究に加えて、米国で臨床試験中の GSK3 阻害剤 9-ING-41 を開発した Actuate 社と共同で治療耐性膵がんの前臨床試験を計画した。
２．膵がんの抗がん剤耐性獲得に伴う悪性形質の解析研究
膵がんで汎用されているゲムシタビン（GEM）に対する耐性獲得は、膵がん治療の未解明医療
ニーズである。そこで、GEM 感受性膵がん BxPC-3 細胞から段階的に GEM 耐性を獲得させた 3
種のクローン細胞株（Anticancer Drug 2015;26:90, 北里大から供与）を対象に、GEM 耐性獲得の生物学的特性を検討した。その結果、耐性獲得に伴い腫瘍浸潤とがん幹細胞形質が増強した。
また、最耐性細胞株をマウス膵内に移植すると、高度の局所浸潤と肝や腹膜転移をきたし、GEM耐性を獲得した膵がん患者の病態を模倣する所見を呈した。さらに、GSK3 がこれらの悪性形
質のハブとして作用することを見出した（論文作成中）。ついで、耐性細胞株の遺伝子発現解析
により、ある種の cyclin D 亜型をはじめ複数の新たな耐性獲得に関わる候補責任分子群を見出した。現在、個々の分子について解析を進めている。
３．ヒト消化管がん組織バンクを中心とする大腸がんの分子病理学的研究
消化管がん研究や臨床研究の基礎資源として 2008 年から本事業を開始し、2010 年にこの事
業を当研究所ヒトがん組織バンクに継承し現在に至っている。この組織資源の共同利用促進のために、日本医療研究開発機構ゲノム医療支援サイト（http://www.biobank.amed.go.jp/biobank/index. html）に情報公開している。帝京大学：竹田 扇らが山梨大学で開発した大気圧イオン化法－質量分析を用いて、大腸がん質量分析診断法開発の共同研究を継続している。大腸組織の質量分析パターンをもとに特有の統計解析と機械学習を組合わせて非がん/がんの判別（診断）アルゴリズムを構築し、90%以上の感度と特異度による判別を可能にした（論文作成予定）。現在、山梨大学：吉村らと島津製作所基盤技術研究所との共同で、大腸がんの質量分析－内視鏡診
断法の内視鏡デバイス開発を進めている。組織バンク検体を共用して、名古屋市立大学（十二指
腸腺腫，早期がん）や香川大学（がんテロメア解析）と共同研究を継続している。そして、香川大
学：松田（故人）が主導する多施設共同研究で、hTERT の第 249 スレオニンのリン酸化と、がん細胞とがん随伴線維芽細胞におけるテロメア長が主要がん種の悪性度と相関し、患者生存期間と逆相関することを明らかにした。

-7-7-

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野【研究費】 （2022 年 1 月以降の新規，継続，分担と連携を含む外部資金の獲得状況）
研究種目・期間（課題番号）

研究代表者

2022年－2024年度
科学研究費補助金
源 利成（基盤研究Ｂ）（22H03144）

研究分担者
研究課題
連携研究者

宮下知治

2022年－2024年度
科学研究費補助金
堂本貴寛（基盤研究Ｃ）（22K07227）
2020年－2022年度
科学研究費補助金
宮下知治（基盤研究Ｃ）（20K09100）

研究経費

直接経費
治療耐性膵がんの悪性形質を繋ぐ
13,400,000 円
分子経路の解明と耐性制御法開発
間接経費
への応用
4,020,000 円
直接経費
抗がん剤耐性獲得膵がん細胞にお3,100,000 円
ける悪性形質連関の解明と治療法
間接経費
開発
930,000 円

太田哲生，
源 利成，
ほか

直接経費
GSK3を基軸とした食道発癌機構の3,200,000 円
解明と新規治療戦略の開発
間接経費
960,000 円

2022年度金沢大学
がん進展制御研究 吉村健太郎 源 利成
所共同研究（一般）

質量分析と機械学習を用いたハイス
ループット大腸がん診断システムの構築

350,000 円

2022年度金沢大学
がん進展制御研究 水津 太
所共同研究（若手）

源 利成

テロメアを介したオートファジー制御と発癌機構の解明

350,000 円

2022年度金沢大学
がん進展制御研究 宮下知治
所共同研究

源 利成

膵癌微小環境内の腫瘍関連線維芽細胞の改変に着目した新規治療法の開発

350,000 円

2022年度金沢大学
がん進展制御研究 佐々木泰史
所共同研究（一般）

澤田 武，
源 利成，
ほか

非乳頭部十二指腸腫瘍におけるERBB受容体ファミリーの解析と治療
標的の探索（一般）

300,000 円

2022年度金沢大学
がん進展制御研究 北村浩一
所共同研究（若手）

松下一之，
源 利成，
ほか

消化器・難治がんのリボソーム生合成の新規メカニズム解明と診断，治療法への応用（若手）350,000 円

2022年度金沢大学
澤田 武，
がん進展制御研究 久保田英嗣 源 利成，所共同研究（一般）ほか

大腸癌における循環腫瘍DNAを用いた抗EGFR抗体薬耐性の検出と病状モニタリングの確立（一般）

300,000 円

2022年度金沢大学
がん進展制御研究 石渡俊行
所共同研究（一般）

スフェア形成法を用いた膵癌幹細胞に有効な薬剤の探索

300,000 円

期間の総額（間接経費を含む）

27,910,000 円

源 利成，
ほか

-8-

研究のあゆみと業績 2022

【研究業績】
Ⅰ．論文発表 [Impact factor 2021]
・英文総説，著書なし
・英文原著

1. Matsuda Y, Ye J, Yamakawa K, Mukai Y, Azuma K, Wu L, Masutomi K, Yamashita T,
Daigo Y, Miyagi Y, Yokose T, Oshima T, Saito H, Morinaga S, Kishida T, Minamoto
T, Kojima M, Kaneko S, Haba R, Kontani K, Kanaji N, Okano K, Muto-Ishizuka M,
Yokohira M, Saoo K, Imaida K, Suizu F. Association of longer telomere length in cancer
cells and cancer-associated fibroblasts with worse prognosis. J Natl Cancer Inst, 2022
Dec 26:djac226. doi: 10.1093/jnci/djac226. Online ahead of print. [11.816]
2. Tran CP, Scurr M, O’Connor L, Buzzelli JN, Ng GZ, Chung Nien Chin S, Stamp LA,
Minamoto T, Giraud AS, Judd LM, Sutton P, Menheniott TR. IL-33 promotes gastrictumour growth in concert with activation and recruitment of inflammatory myeloid cells.
Oncotarget 13: 785-99, 2022. doi: 10.18632/oncotarget.28238 [5.168]
3. Matsuda Y, Yamashita T, Ye J, Yasukawa M, Yamakawa K, Mukai Y, Machitani M, Daigo
Y, Miyagi Y, Yokose T, Oshima T, Ito H, Morinaga S, Kishida T, Minamoto T, Yamada
S, Takei J, Kaneko MK, Kojima M, Kaneko S, Masaki T, Hirata M, Haba R, Kontani K,
Kanaji N, Miyatake N, Okano K, Kato Y, Masutomi K. Phosphorylation of hTERT at
threonine 249 is a novel tumor biomarker of aggressive cancer with poor prognosis in
multiple organs. J Pathol 257 (2): 172-85, 2022. doi: 10.1002/path.5876 [9.883]
4. Kondo H, Mishiro K, Iwashima Y, Qiu Y, Kobayashi A, Lim K, Domoto T, Minamoto
T, Ogawa K, Kunishima M, Hazawa M, Wong, RW. Discovery of a novel aminocyclopropenone compound that inhibits BRD4-driven nucleoporin NUP210 expression andattenuates colorectal cancer growth. Cells 11 (3): 317, 2022. doi: 10.3390/cells11030317[7.666]
Ⅱ．学会発表・国際学会

1. Takahiro Domoto, Masahiro Uehara, Osamu Takeuchi, Tomoharu Miyashita, Toshinari
Minamoto. GSK3 interconnects tumor invasion and stemness in pancreatic cancer
acquiring resistance to gemcitabine. The 7th JCA-AACR Special Joint Conference: The
Latest Advances in Pancreatic Cancer Research: From Basic Science to Therapeutics,
June 8th (Wed)~10th (Fri), 2022, Kyoto, Japan.
2. Masahiro Uehara, Takahiro Domoto, Satoshi Takenaka, Dilireba Bolidong, Osamu
Takeuchi, Tomoharu Miyashita, Toshinari Minamoto. Glycogen synthase kinase-3
participates in acquired resistance to gemcitabine in pancreatic cancer. The 7th JCAAACR Special Joint Conference: The Latest Advances in Pancreatic Cancer Research:
From Basic Science to Therapeutics, June 8th (Wed)~10th (Fri), 2022, Kyoto, Japan.
・国内（全国）学会

3. 上原将大，堂本貴寛，ディリラバ ボリドン，宮下知治，源 利成．GSK3 は膵がんのゲムシタ
ビン獲得耐性に寄与する．第 31 回日本癌病態治療研究会，2022 年 6 月 23 日（木），24 日-9-9-

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野（金），鳴門市．

4. Takahiro Domoto, Satoshi Takenaka, Masahiro Uehara, Tatsuhiko Furukawa, Tomoharu
Miyashita , Toshinari Minamoto. GSK3β participates in malignant properties ofpancreatic cancer acquiring chemoresistance. 堂本貴寛，竹中 哲，上原将大，古川龍彦，

宮下知治，源 利成．GSK3 は薬剤耐性獲得膵がんの悪性進展に寄与する．第 81 回日本
癌学会学術総会，2022 年 9 月 29 日（木）-10 月 01 日（土），パシフィコ横浜，横浜市．

5. Hiroki Sato, Katsuya Sakai, Ryu Imamura, Toshinari Minamoto, Kunio Matsumoto.
Metastatic platform formation triggered by distal HGF conversion. 佐藤拓輝，酒井克也，

今村 龍，源 利成，松本邦夫．HGF 活性の遠隔制御によるがん転移ニッチ形成．第 81 回日本癌学会学術総会，2022 年 9 月 29 日（木）-10 月 01 日（土），パシフィコ横浜，横浜市．

6. Yoko Matsuda, Taro Yamashita, Juanjuan Ye, Keiko Yamakawa, Yuri Mukai, Toshinari
Minamoto, Yukinari Kato, Kenkichi Masutomi, Futoshi Suizu. Phosphorylation ofhTERT at threonine 249 is a novel biomarker of aggressive cancer with poor prognosis.
松田陽子，山下太郎，葉 娟娟，山川けいこ，向井裕理，源 利成，加藤幸成，増富健吉，水津太．ヒトテロメラーゼ逆転写酵素のスレオニン 249 のリン酸化は予後不良な高悪性度腫瘍の新規マーカーである．第 81 回日本癌学会学術総会，2022 年 9 月 29 日（木）-10 月 01 日（土），パシフィコ横浜，横浜市．

7. 上原将大，堂本貴寛，堀家慎一，Francis Giles, Ludimila Cavalcante，高橋智聡，源 利成．膵がんの抗がん剤獲得耐性に対する臨床開発 GSK3 阻害剤と承認済 CDK4/6 阻害剤の効
果．第 33 回日本消化器癌発生学会総会，シンポジウム 2 『臨床応用を意識した消化器癌研究』，2022 年 11 月 11 日（金），12 日（土），一橋大学一橋講堂，東京．

8. 堂本貴寛，上原将大，宮下知治，源 利成．GSK3 は膵がんの獲得耐性と浸潤能，がん幹細胞性を統合する．第 33 回日本消化器癌発生学会総会，シンポジウム 3 『多様性を持った癌細胞の制圧』，2022 年 11 月 11 日（金），12 日（土），一橋大学一橋講堂，東京．

9. 橋本明史，宮下知治，島崎猛夫，源 利成，高村博之．バレット食道から発癌過程での微小環境の変化と GSK3 阻害による抑制効果．第 33 回日本消化器癌発生学会総会，ワークショッ
プ１（食道），2022 年 11 月 11 日（金），12 日（土），一橋大学一橋講堂，東京．

・司会・座長など

10. 源 利成．司会 一般演題４ 蛋白・代謝産物．第 42 回日本分子腫瘍マーカー研究会，2022
年 9 月 28 日（水），オンライン開催．

04-1 辻尾 元，丸尾晃司，山本百合恵，瀬良知央，杉本敦史，笠島裕明，三木友一朗，吉井
真美，田村達郎，豊川貴弘，田中浩明，六車一哉，大平雅一，八代正和．クラスター分
析による胃癌リンパ節転移および予後に関与する蛋白および遺伝子の解析．

04-2 平野秀和，阿部雄一，野島陽水，青木雅彦，庄司広和，磯山純子，本田一文，朴 成和，
水口賢治，朝長 毅，足立 淳．Stage IV 胃癌における内視鏡生検検体を用いた経時的
リン酸化プロテオーム解析による新規治療標的の探索．

04-3 菅 元泰，千葉哲博，紺野 亮，中川 良，大山 広，大野 泉，日下部裕子，高橋幸治，永嶌裕樹，三浦義史，大内麻愉，川島祐介，小原 収，加藤直也．高精度質量分析を用いた胆汁プロテオーム解析の胆道癌の新規バイオマーカー探索における有用性の検討．

04-4 野口 玲，近藤 格．EGFR 感受性遺伝子変異を有する進行非小細胞肺がんにおける予後予測キナーゼの同定：三次元ペプチドアレイによる網羅的キナーゼ活性解析を用いて．

- 10 -

研究のあゆみと業績 202204-5 軍司大悟，阿部雄一，長山 聡，坂井義治，小濵和貴，朝長 毅，足立 淳．リン酸化プロ
テオームデータと薬剤感受性データの統合解析による薬剤耐性大腸がん肝転移巣の新規治療標的探索．

04-6 山道 岳，加藤大悟，明庭昇平，岡 利樹，奥田洋平，植村俊彦，山本顕生，冨山栄輔，
石津谷祐，山本致之，波多野浩士，河嶋厚成，植村元秀，野々村祝夫．前立腺癌における GDF15 プロペプチドの骨転移診断マーカーとしての有用性．
・その他（講演，社会・地域貢献を含む）

11. 源 利成．大腸がんのしくみと医療を一緒に学びましょう．金沢大学公開講座：がん医療の最前線，2022 年 6 月 11 日（土），金沢大学西町サテライト・プラザ，金沢市．

12. 源 利成．膵がん難治性の細胞特性 ― その仕組みと私たちの取りくみ ―．第 38 回日本臨床細胞学会北陸連合会学術集会，2022 年 9 月 4 日（日），金沢（ウェブ）．

Ⅲ．学会開催該当なし

Ⅳ．
．究成果による知的財産権の出願状況
該当なし
Ⅴ．
．新聞，報道など
該当なし- 11 - 11 -

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野【附記１】 金沢大公開講座（抜粋）- 12 -

研究のあゆみと業績 2022

【附記２】 金沢大がん研 News Letter（（抜粋）：共同研究者紹介※金沢大学がん進展制御研究所共同利用・共同研究拠点の承諾を得て転載。

- 13 - 13 -

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野- 14 -

研究のあゆみと業績 2022

- 15 - 15 -

金 沢大学がん進 展制御 研究所 腫瘍 制御 (旧： 遺伝子 診断 ) 研 究分野【附記３】 学位論文： 2022 年 3 月および 12 月修了
Ota R, Sawada T, Tsuyama S, Yao T, Sasaki Y, Suzuki H, Kaizaki Y,
Hasatani K, Yamamoto E, Nakanishi H, Inagaki S, Tsuji S, Yoshida N,
Doyama H, Kasashima S, Kubota E, Kataoka H, Tokino T, Minamoto T.
Integrated genetic and epigenetic analysis of cancer-related genes in nonampullary duodenal adenomas and intramucosal adenocarcinomas. J
Pathol 252 (3): 330-42, 2020. doi: 10.1002/path.5529.
The molecular and clinical characteristics of non-ampullary duodenal adenomas and intramucosal
adenocarcinomas are not fully understood because they are rare. To clarify these characteristics, we
performed genetic and epigenetic analysis of cancer-related genes in these lesions. One hundred andseven non-ampullary duodenal adenomas and intramucosal adenocarcinomas, including 100 small
intestinal-type tumors (90 adenomas and 10 intramucosal adenocarcinomas) and 7 gastric-type
tumors (2 pyloric gland adenomas and 5 intramucosal adenocarcinomas), were investigated. Using
bisulfite pyrosequencing, we assessed the methylation status of CpG island methylator phenotype(CIMP) markers and MLH1. Then using next-generation sequencing, we performed targeted exome
sequence analysis within 75 cancer-related genes in 102 lesions. There were significant differencesin the clinicopathological and molecular variables between small intestinal- and gastric-type tumors,which suggests the presence of at least two separate carcinogenic pathways in non-ampullary
duodenal adenocarcinomas. The prevalence of CIMP-positive lesions was higher in intramucosal
adenocarcinomas than in adenomas. Thus, concurrent hypermethylation of multiple CpG islands islikely associated with development of non-ampullary duodenal intramucosal adenocarcinomas.
Mutation analysis showed that APC was the most frequently mutated gene in these lesions (56/102;55%), followed by KRAS (13/102; 13%), LRP1B (10/102; 10%), GNAS (8/102; 8%), ERBB3(7/102; 7%), and RNF43 (6/102; 6%). Additionally, the high prevalence of diffuse or focal nuclear
β-catenin accumulation (87/102; 85%) as well as mutations of WNT pathway components (60/102;59%) indicates the importance of WNT signaling to the initiation of duodenal adenomas. The higher
than previously reported frequency of APC gene mutations in small bowel adenocarcinomas as wellas the difference in the APC mutation distributions between small intestinal-type adenomas andintramucosal adenocarcinomas may indicate that the adenoma-carcinoma sequence has only limited
involvement in duodenal carcinogenesis.

Kotake M, Bando H, Kaneko M, Takemura H, Minamoto T, Kawakami
K. LOH of the thymidylate synthase locus in combination with genotype
has prognostic and predictive significance in colorectal cancer. Mol Clin
Oncol 15 (5): 235, 2021. doi: 10.3892/mco.2021.2398.
The aim of the current study was to investigate the prognostic and predictive
significance of polymorphisms in the thymidylate synthase (TS) gene, alongside theloss of heterozygocity (LOH) at this gene locus in patients with colorectal cancer. Genotyping was
carried out for a variable number tandem repeat (VNTR) polymorphism in the TS 5'-untranslated
region, a G/C single nucleotide polymorphism (SNP) located within this VNTR, and for TS LOH
status in 246 colorectal cancer and paired normal DNA samples. The results were analyzed in relation
to clinicopathological features, including the prognostic and predictive significance of TS genotype
in patients who underwent curative surgery. Complete VNTR, SNP and LOH information for TS was
obtained in 226 cases. No significant associations were observed between normal tissue TS genotype
status and clinicopathological features. LOH of TS was observed in 58% of tumor samples and was
associated with poor prognosis independently of clinical stage. Cases exhibiting TS LOH wereclassified into the three groups of 2R/loss, 3G/loss and 3C/loss. Patients with 3C/loss genotype status
had poor outcomes when treated by surgery alone, but their survival was similar to patients with othergenotypes following Fluorouracil (5-FU)-based adjuvant chemotherapy. The results suggested that
LOH of the TS locus may be a significant prognostic factor in colorectal cancer, with the genotypeof the residual allele also demonstrating an influence on prognosis. In conclusion, LOH status should
be considered when TS genotype is explored as a potential prognostic and predictive marker for 5FU-based adjuvant chemotherapy in colorectal cancer.

- 16 -

Journal of Pathology
J Pathol November 2020; 252: 330–342
Published online 12 September 2020 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.5529

ORIGINAL PAPER

Integrated genetic and epigenetic analysis of cancer-related genes in
non-ampullary duodenal adenomas and intramucosal
adenocarcinomas
Ryosuke Ota1†, Takeshi Sawada1,2†* , Sho Tsuyama3, Yasushi Sasaki4, Hiromu Suzuki5 , Yasuharu Kaizaki6,
Kenkei Hasatani7, Eiichiro Yamamoto5, Hiroyoshi Nakanishi1, Satoko Inagaki8, Shigetsugu Tsuji9, Naohiro Yoshida9,
Hisashi Doyama9, Hiroshi Minato10, Keishi Nakamura11, Satomi Kasashima12, Eiji Kubota2, Hiromi Kataoka2,
Takashi Tokino13, Takashi Yao3 and Toshinari Minamoto1
1

Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan3
Department of Human Pathology, Juntendo University Graduate School of Medicine, Tokyo, Japan
4
Division of Biology, Department of Liberal Arts and Sciences, Center for Medical Education, Sapporo Medical University, Sapporo, Japan
5
Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan
6
Department of Pathology, Fukui Prefectural Hospital, Fukui, Japan
7
Department of Gastroenterology, Fukui Prefectural Hospital, Fukui, Japan
8
Department of Advanced Research in Community Medicine, Kanazawa University Graduate School of Medical Sciences, Kanazawa, Japan
9
Department of Gastroenterology, Ishikawa Prefectural Central Hospital, Kanazawa, Japan10
Department of Pathology, Ishikawa Prefectural Central Hospital, Kanazawa, Japan
11
Department of Gastroenterological Surgery, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
12
Department of Clinical Laboratory Science, Kanazawa University, Kanazawa, Japan
13
Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University, Sapporo, Japan
2*Correspondence to: T Sawada, Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences,
1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail: takeshisawada@icloud.com†

These authors contributed equally to this work.

Abstract
The molecular and clinical characteristics of non-ampullary duodenal adenomas and intramucosal adenocarcinomas
are not fully understood because they are rare. To clarify these characteristics, we performed genetic and epigenetic
analysis of cancer-related genes in these lesions. One hundred and seven non-ampullary duodenal adenomas andintramucosal adenocarcinomas, including 100 small intestinal-type tumors (90 adenomas and 10 intramucosal adenocarcinomas) and 7 gastric-type tumors (2 pyloric gland adenomas and 5 intramucosal adenocarcinomas), were
investigated. Using bisulﬁte pyrosequencing, we assessed the methylation status of CpG island methylator phenotype(CIMP) markers and MLH1. Then using next-generation sequencing, we performed targeted exome sequence analysis
within 75 cancer-related genes in 102 lesions. There were signiﬁcant differences in the clinicopathological andmolecular variables between small intestinal- and gastric-type tumors, which suggests the presence of at least
two separate carcinogenic pathways in non-ampullary duodenal adenocarcinomas. The prevalence of CIMP-positive
lesions was higher in intramucosal adenocarcinomas than in adenomas. Thus, concurrent hypermethylation of multiple CpG islands is likely associated with development of non-ampullary duodenal intramucosal adenocarcinomas.
Mutation analysis showed that APC was the most frequently mutated gene in these lesions (56/102; 55%), followed
by KRAS (13/102; 13%), LRP1B (10/102; 10%), GNAS (8/102; 8%), ERBB3 (7/102; 7%), and RNF43 (6/102; 6%).
Additionally, the high prevalence of diffuse or focal nuclear β-catenin accumulation (87/102; 85%) as well as mutations of WNT pathway components (60/102; 59%) indicates the importance of WNT signaling to the initiation ofduodenal adenomas. The higher than previously reported frequency of APC gene mutations in small bowel adenocarcinomas as well as the difference in the APC mutation distributions between small intestinal-type adenomas andintramucosal adenocarcinomas may indicate that the adenoma–carcinoma sequence has only limited involvementin duodenal carcinogenesis.
© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of GreatBritain and Ireland.

Keywords: non-ampullary duodenal adenoma; small bowel adenocarcinoma; methylation; CpG island methylator phenotype; mutation
Received 2 March 2020; Revised 20 July 2020; Accepted 28 July 2020

No conﬂicts of interest were declared.

© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use anddistribution in any medium, provided the original work is properly cited, the use is non-commercial and no modiﬁcations or adaptations are made.

- 17 -

Mutation and methylation in duodenal intramucosal neoplasia

Introduction
Small intestinal cancers are rare. They comprise only 2–
3% of the total annual cancer incidence in the digestivesystem [1,2], and a recent epidemiological study in the
Unites States of America showed that the age-adjusted
incidence rate (IR) for small intestinal cancers is only
2.10/100 000 person-years [3]. These small intestinal
cancers include neuroendocrine cancers (IR = 0.83),
carcinomas (IR = 0.66), sarcomas (IR = 0.20), and lymphomas (IR = 0.38), with small bowel adenocarcinomas(SBAs) accounting for approximately 69% of the carcinomas. The incidence of carcinomas is most prominent
in the duodenum, and duodenal carcinomas haveincreased more markedly than other small intestinal cancers []. Duodenal adenomas are also uncommon lesions,with a reported prevalence of less than 0.1–0.3% inpatients undergoing upper gastrointestinal endoscopy[4,5]. Nonetheless, with the advent of surveillanceendoscopy and improvements in endoscopic imaging,these lesions are now being detected incidentally [6].
Moreover, these lesions are thought to progress into duodenal adenocarcinomas (DAs), via the adenoma–
carcinoma sequence [7–9], a common pathway for colorectal cancer (CRC) development [10]. However, theirclinicopathological characteristics and natural course
have not been investigated in detail, due to their rarity.
Because SBAs have a signiﬁcantly poorer prognosis
than CRCs [11], early detection and treatment are crucial. In particular, preoperative diagnosis to distinguishlesions that should be followed up from those that
require treatment is an important problem [6], andmolecular characterization of premalignant duodenal
lesions is essential to address this issue.
Concurrent methylation of multiple CpG islands(CGIs) was ﬁrst characterized as CpG island methylator
phenotype (CIMP) in CRC by Toyota et al [12]. Since
then, CIMP has been reported in various types of tumors[13]. Multiple studies also reported the presence of
CIMP in SBAs, including DAs [14,15], and the methylation proﬁles of DAs reportedly differ from biliary andampullary carcinomas [14]. Moreover, CIMP positivity,
albeit without MLH1 methylation, is reportedly associated with a poor prognosis in DA patients [15]. An earlier study analyzed DNA methylation in ampullary andnon-ampullary duodenal adenomas, but that study did
not analyze these tumor types separately [16]. Thus, epigenetic alterations in early non-ampullary duodenal
lesions remain to be elucidated.
By contrast, genetic analyses of SBAs, including nonampullary DAs, have been performed by multiple
groups [17–23]. Schrock et al [20] analyzed mutationsof cancer-related genes in 317 SBA samples and showedthat the genetic signatures of SBAs were distinct fromthose in CRCs or gastric cancers. In addition, exome
sequencing revealed potential driver genes, dysregulated
oncogenic pathways, and targetable mutations in SBAs
[18,19,21,22]. Still, the genetic alterations in early duodenal lesions are not fully understood, with only one

331

earlier study analyzing mutations of 50 cancer-related
genes in a limited number of samples [24].
To gain further insight into duodenal carcinogenesis,
we analyzed the methylation and mutation status of alarge number of non-ampullary duodenal adenomas
and intramucosal adenocarcinoma samples, andassessed their clinicopathological signiﬁcance. We also
evaluated the involvement of the WNT signaling pathway, which is reportedly dysregulated in non-ampullary
duodenal adenomas and adenocarcinomas [25–27].

Materials and methods
Patients and tissue samples
Specimens of non-ampullary duodenal adenomas andintramucosal adenocarcinomas (n = 107) were obtained
from 107 Japanese patients who underwent endoscopic
mucosal resection or endoscopic submucosal dissection at Ishikawa Prefectural Central Hospital or Fukui
Prefectural Hospital between 2008 and 2019. Normal
samples were also obtained from normal-appearing adjacent mucosa in 15 patients with small intestinal-type
duodenal adenomas included in the present study.
Patients with hereditary cancer syndrome such as familial adenomatous polyposis (FAP) or Peutz–Jeghers syndrome, as well as those with inﬂammatory bowel
diseases, such as Crohn’s disease, were excluded. Information on their BMI and smoking status at the time oftreatment was also obtained. Approval of this study
was obtained from the Institutional Review Board of
Ishikawa Prefectural Central Hospital and Fukui Prefectural Hospital, Kanazawa University, Juntendo University, and Sapporo Medical University.

Endoscopic analysis
High-resolution magnifying endoscopes (GIF-H290Z;
Olympus, Tokyo, Japan) were used for all upper gastrointestinal endoscopic analyses. The morphology of duodenal lesions was determined according to the Parisclassiﬁcation [28]. All lesions detected during esophagogastroduodenoscopy were observed at high magniﬁcation using narrow band imaging, after which samples
were treated by endoscopic resection for histologicalanalysis. Locations within the duodenum were subcategorized into bulb, descending part, and transverse part.
Ninety-ﬁve small intestinal-type tumors, of which
detailed information about location was available, werealso subcategorized into tumors within the proximal part(from bulb to periampulla) and those within the distal
part (from periampulla to transverse part), as described
previously [26].

Histological analysis
Histological studies were ﬁrst carried out at Ishikawa
Prefectural Central Hospital and Fukui Prefectural Hospital. The histological ﬁndings for all specimens were

© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
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332

R Ota, T Sawada et althen re-reviewed by two independent pathologists(ST and TY) blinded to the clinical and molecular information. The presence of small intestinal phenotypes wasdetermined based on the presence of a brush border, goblet cells, and Paneth cells in H&E-stained specimens.
Immunohistochemical staining for CD10, MUC2,
MUC5AC, and MUC6 was performed as described previously [29]. Small intestinal-type tumors were deﬁned
by CD10 and MUC2 staining. The presence of gastrictype differentiation was deﬁned by MUC5AC andMUC6 staining. Tumors with the small intestinal phenotype were classiﬁed as small intestinal-type low-grade
adenomas (SLAs), small intestinal-type high-grade adenomas (SHAs), or small intestinal-type intramucosal
adenocarcinomas (SCAs) according to the World HealthOrganization (WHO) tumor classiﬁcation system (supplementary material, Figure S1) [8]. Tumors with thegastric phenotype were classiﬁed as pyloric gland adenomas (PGAs) or gastric-type intramucosal adenocarcinomas (GCAs). SLAs and PGAs were classiﬁed as
category 3 tumors, while SHAs, SCAs, and GCAs wereclassiﬁed as category 4 tumors according to the revised
Vienna classiﬁcation of gastrointestinal epithelial neoplasia [30]. SCAs correspond to non-invasive carcinomas (category 4.2), suspicious for invasive carcinomas(category 4.3), or intramucosal carcinomas (category
4.4). The clinicopathological features of the lesions are
summarized in Table 1.

DNA isolation
DNA was isolated from formalin-ﬁxed, parafﬁnembedded (FFPE) sections using a QIAamp DNA FFPE
Tissue kit (Qiagen, Hilden, Germany). A TaqMan
RNase P Detection Reagents kit (Thermo Fisher Scientiﬁc, Waltham, MA, USA) was used to quantify the
puriﬁed DNA.

DNA methylation analysis
DNA methylation was analyzed using bisulﬁte pyrosequencing as described previously [31]. A cut-off valueof 15% was used to deﬁne genes as methylationpositive. We used ﬁve CIMP markers (CACNA1G,
IGF2, NEUROG1, RUNX3, and SOCS1) proposed by
Weisenberger et al [32]. Tumors were deﬁned as
CIMP-positive if two or more loci showed methylation and as CIMP-high (CIMP-H) if three or more locishowed methylation, as previously reported [16]. In
addition, methylation of the MLH1 gene was investigated. Primer sequences are shown in supplementarymaterial, Table S1.

Targeted amplicon sequencing analysis
A customized panel, encompassing all exons for 75 cancer-related genes including those frequently mutated in
SBAs [17–21,33,34], was created using the Ion AmpliSeq Designer (Thermo Fisher Scientiﬁc) (supplementarymaterial, Table S2). Genes whose mutations had beenreported in duodenal adenomas were also included[24]. The assay design consisted of 3663 amplicons withan average length of 112 bp, covering 95.5% of the
366 kb target sequence. Library preparation andsequencing with an Ion Proton sequencer were performed as described previously [35,36]. The templates
were sequenced after emulsion PCR with 12–16 samples
per Ion PI chip using an Ion PI HI-Q Chef kit (Thermo
Fisher Scientiﬁc).

Identiﬁcation of somatic mutations and copy
number variations
Somatic mutations and copy number variations (CNVs)
were detected as described previously [35]. Human
genome build 19 (hg19) was used as a reference. Signal
processing, base-calling, mapping to the hg19 reference,alignment, and further quality ﬁltering were performed
using Torrent Suite version 5.0 (Thermo Fisher Scientiﬁc). Somatic mutations, including point mutations,insertions, and deletions, were detected using Ion
Reporter Software 5.0 (Thermo Fisher Scientiﬁc).
Because matched normal controls were not available,
we utilized Ion Reporter software tumor–normal workﬂow using Demo AmpliSeq Exome control as the normal control for excluding common single nucleotide
polymorphisms (SNPs). A sequencing coverage of 20x
and a minimum variant frequency of 5% of the total
number of distinct tags were used as cut-offs. The pathogenic status of the variant was stated if it was a missense variant with less than 0.1% global minor allele
frequency in the dbSNP and/or the variant was suggested
as being pathogenic in the ClinVar, COSMIC, SIFT, or
PolyPhen-2 databases. Variants with allele frequencies
between 0.4 and 0.6 or greater than 0.9 were considered
germline variants unless listed as a pathogenic variant.
Integrative Genomics Viewer (IGV) software (http://
software.broadinstitute.org/software/igv/) was used toﬁlter out possible strand-speciﬁc errors, such as a mutation that was identiﬁed in the forward or reverse DNA
strand but not in both strands. CNVs were also detectedusing Ion Reporter Software with an algorithm based on
the Hidden Markov Model. Recurrent genomic regionswith CNVs were detected using copy numbers greaterthan 3 and less than 1 for gains and losses, respectively.

Immunohistochemistry
Immunohistochemical studies of β-catenin expression
were performed in 102 samples, as described previously[37]. A mouse anti-β-catenin monoclonal antibody(1:1000 dilution, Clone 14; BD Biosciences, San Jose,
CA, USA) was used. Expression of β-catenin was evaluated semi-quantitatively in tumor cells with β-cateninpositive nuclei, as reported previously [38]: negative,
0–9%; focal, 10–49%; and diffuse, > 50%. All slides
were evaluated by two pathologists (ST and TY) blinded
to the clinical and molecular data. In addition, immunohistochemical studies of two mismatch repair (MMR)
proteins, MLH1 (1:1000 dilution, Ab92312; Abcam,
Cambridge, UK) and MSH2 (1:1000 dilution, Ab© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 19 -J Pathol 2020; 252: 330–342
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Mutation and methylation in duodenal intramucosal neoplasia

333

Table 1. Clinicopathological features of non-ampullary duodenal lesions.
Histology
Vienna classiﬁcation

Patients’ characteristics (n)Age (years, mean  SD)GenderMale, n (%)
Female, n (%)
BMI (kg/m2, mean  SD)Smoking status
Current smoker, n (%)
Former/non-smoker, n (%)
Lesion characteristics
Location in duodenum
Bulb, n (%)
Descending part, n (%)
Transverse part, n (%)
Others, n (%)
Lesion size (mm, mean  SD)Morphology
Protruding (0-Ip, 0-Is, 0-Isp), n (%)
Mixed (0-I + IIa), n (%)
Nonprotruding and nonexcavated(0-IIa, 0-IIc, 0-IIa + IIc), n (%)
N/A, n (%)

Total

SLA
SHA
SCA
Category 3
Category 4
Small intestinal type

PGA
GCA
Category 3
Category 4
Gastric type

P value(Small intestinal typeversus gastric type)107
64  103264  958
63  101066  12269  3578  480 (75%)27 (25%)22.9 3.4

24 (75%)8 (25%)
23.2  3.543 (74%)15 (26%)22.4  3.58 (80%)
2 (20%)
24.4  2.41 (50%)
1 (50%)
22.2  1.14 (80%)
1 (20%)
23.7  3.2> 0.9999

29 (27%)78 (73%)7 (22%)
25 (78%)20 (34%)38 (66%)0 (0%)
10 (100%)

0 (0%)
2 (100%)2 (40%)
3 (60%)> 0.9999

17 (16%)
86 (80%)3 (3%)
1 (1%)
13  84 (13%)
27 (84%)1 (3%)
0 (0%)
11  66 (10%)
49 (85%)2 (3%)
1 (2%)
14  8

1 (10%)
9 (90%)
0 (0%)
0 (0%)
19  10

2 (100%)0 (0%)
0 (0%)
0 (0%)
10  04 (80%)
1 (20%)
0 (0%)
0 (0%)
18  5< 0.0001(Bulb versus non-bulb)

16 (15%)5 (5%)

4 (13%)
0 (0%)

4 (7%)
3 (5%)

3 (30%)
1 (10%)

1 (50%)
1 (50%)

4 (80%)
0 (0%)

0.0002(Protruding or mixed
versus nonprotruding)

85 (79%)28 (87%)50 (86%)6 (60%)

0 (10%)

1 (20%)

1 (1%)

0 (0%)

1 (2%)

0 (0%)

0 (0%)

0 (0%)

0.0022

0.79

0.41

GCA, gastric-type intramucosal adenocarcinoma; N/A, not available; PGA, pyloric gland adenoma; SCA, small intestinal-type intramucosal adenocarcinoma; SHA, small
intestinal-type high-grade adenoma; SLA, small intestinal-type low-grade adenoma.

227 841; Abcam), was performed in 107 samples toassess microsatellite instability (MSI) status. To evaluate
expression, lymphocytes in adjacent normal tissue wereused as an internal positive control. When nuclear staining was identiﬁed in epithelial cells, the lesion wasdeﬁned as positive for MMR proteins. All slides were
evaluated by a pathologist (SK) blinded to the clinicaland molecular data.

Statistical analysis
Continuous data were analyzed using t-tests (for twogroups) or ANOVA with a post hoc Tukey’s HSD test(for more than two groups). Comparison of categorical
data between two or more groups was performed using
the Fisher’s exact test or chi-squared test. Values ofp < 0.05 were considered statistically signiﬁcant. All
statistical analyses were performed using GraphPad
Prism 8 (GraphPad Software, San Diego, CA, USA).

Results
Clinicopathological characteristics of non-ampullary
duodenal lesions
The clinicopathological and molecular characteristics of
107 non-ampullary duodenal lesions analyzed in thisstudy are summarized in Table 1. The average age atendoscopic treatment was 64 years. The male-to-female
ratio was approximately 3:1, which is consistent withprevious studies from Japan [9,26,39]. When compared
between cases with small intestinal-type (SLA, SHA,
and SCA) and gastric-type (PGA and GCA) tumors,
age at treatment was signiﬁcantly higher in those withgastric-type tumors (t-test, p = 0.0022). In addition,
tumors with the gastric phenotype were more prevalent
in the duodenal bulb (6/7, 86%) than were those withthe small intestinal phenotype (11/100, 11%) (Fisher’s
exact test, p < 0.0001). On endoscopic observation,
most tumors with gastric differentiation were protruding
lesions or mixed lesions that included a protruding portion (6/7, 86%), and differed signiﬁcantly from tumors
with small intestinal differentiation, most of which were
non-protruding and non-excavated lesions (84/100,
84%) (Fisher’s exact test, p = 0.0002, Table 1). We also
compared the clinicopathological characteristics ofsmall intestinal-type tumors based on the proximal or
distal locations (supplementary material, Table S3). Signiﬁcant differences were found in the tumor histologicaltypes (SLA, SHA or SCA, chi-squared test, p = 0.017)
and categories (3 or 4, Fisher’s exact test, p = 0.0075)
between tumors in the proximal and distal duodenum.
In addition, the prevalence of nonprotruding and nonexcavated type tumors was signiﬁcantly higher in the distal
part (Fisher’s exact test, p = 0.037).

Methylation analysis of CIMP markers and MLH1
Among non-ampullary duodenal lesions, we assessed
the methylation status of cancer-associated genes in
107 lesions and assessed the CIMP and CIMP-H status© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 20 -J Pathol 2020; 252: 330–342
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R Ota, T Sawada et al

in 106 lesions. Twenty-ﬁve lesions (25/106, 24%) weredeﬁned as CIMP-positive, and seven lesions (7/106,7%) were deﬁned as CIMP-H (Figure 1 and Table 2).
The prevalence of CIMP-positive lesions was signiﬁcantly associated with male gender (Fisher’s exact test,
p = 0.033), older age (cut-off value 75 years, Fisher’s
exact test, p = 0.043), and larger tumor size (cut-off
value 15 mm, Fisher’s exact test, p = 0.032) (Table 3).
By contrast, BMI, smoking status, and the location andendoscopic morphology of the lesions were not associated with CIMP positivity. When samples were divided
into adenomas (SLA, SHA, and PGA) and intramucosal
adenocarcinomas (SCA and GCA), the prevalence of
CIMP-positive lesions was higher in intramucosal

adenocarcinomas (6/15, 40%) than in adenomas(19/91, 21%), although there were no statistical signiﬁcant differences (Fisher’s exact test, p = 0.18, Table 3).
When only small intestinal-type tumors were analyzed,
CIMP positivity was not associated with tumor histological type (SLA, SHA, and SCA). Moreover, the prevalence of CIMP-positive lesions was not associated witheither histological classiﬁcation (small intestinal type or gastric type) or Vienna classiﬁcation (category 3 or 4).
Although two MLH1 methylation-positive lesions weredetected among the SHA samples, methylation levels were
below 20% (15.8% and 16.5%) in these lesions. In addition,
there was no association between MLH1 methylation status
and CIMP or CIMP-H positivity (Figure 1). Although there

Figure 1. Methylation and mutation proﬁles in non-ampullary duodenal adenomas and intramucosal adenocarcinomas. Summarized resultsfor CIMP marker methylation, CIMP status, MLH1 methylation, and BRAF/KRAS/GNAS mutations in tumors with the indicated histologicaltypes are shown. CIMP, CpG island methylator phenotype; GCA, gastric-type intramucosal adenocarcinoma; PGA, pyloric gland adenoma;
SCA, small intestinal-type intramucosal adenocarcinoma; SHA, small intestinal-type high-grade adenoma; SLA, small intestinal-type lowgrade adenoma.
© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 21 -J Pathol 2020; 252: 330–342
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Mutation and methylation in duodenal intramucosal neoplasia

335

Table 2. Molecular characteristics and β-catenin expression of the respective histological types of non-ampullary duodenal lesions.
Histology
Gene mutation (n)APC mutation, n (%)
BRAF mutation, n (%)
KRAS mutation, n (%)
GNAS mutation, n (%)
Epigenetic alteration (n)CIMP*, n (%)
CIMP-high*, n (%)
MLH1 methylation, n (%)β-catenin expression (n)Negative, n (%)
Focal, n (%)
Diffuse, n (%)

Total
102
56 (55%)5 (5%)
13 (13%)
8 (8%)
107
25 (24%)7 (7%)
2 (2%)
102
15 (15%)33 (32%)54 (53%)

SLA
SHA
SCA
Small intestinal type32
17 (53%)
2 (6%)
1 (3%)
0 (0%)
32
5 (16%)
2 (6%)
0 (0%)
32
3 (10%)
11 (34%)18 (56%)58
34 (59%)2 (3%)
4 (7%)
1 (2%)58
14 (24%)3 (5%)
2 (3%)58
5 (8%)
19 (33%)34 (59%)5
3 (60%)
1 (20%)
2 (40%)
1 (20%)
10
3 (30%)
2 (20%)
0 (0%)
5
1 (20%)
2 (40%)
2 (40%)PGA
GCA
Gastric type2
1 (50%)
0 (0%)
2 (100%)1 (50%)2
0 (0%)
0 (0%)
0 (0%)
2
1 (50%)
1 (50%)
0 (0%)

5
1 (20%)
0 (0%)
4 (80%)
5 (100%)53 (60%)
0 (0%)
0 (0%)
5
5 (100%)0 (0%)
0 (0%)

P value(small intestinal type versus gastric type)0.24> 0.9999< 0.0001< 0.0001
0.36> 0.9999> 0.9999< 0.0001(focal or diffuse versus negative)

CIMP, CpG island methylator phenotype; GCA, gastric-type intramucosal adenocarcinoma; PGA, pyloric gland adenoma; SCA, small intestinal-type intramucosal adenocarcinoma; SHA, small intestinal-type high-grade adenoma; SLA, small intestinal-type low-grade adenoma.
β-catenin expression was categorized as negative (0–9%), focal (10–49%), and diffuse (> 50%).
*CIMP status and CIMP-high status were analyzed in 106 lesions.

Table 3. Relationship between CIMP status and clinicopathological characteristics of non-ampullary duodenal lesions.
Characteristics
Patients (n)Age (years, mean  SD)
< 75, n (%)≥ 75, n (%)
GenderMale, n (%)
Female, n (%)
BMI (kg/m2, mean  SD)Smoking status
Current smoker, n (%)
Former/non-smoker, n (%)
Location
Bulb, n (%)
Descending – transverse part, n (%)
Other, n (%)
Size (mm, mean  SD)
< 15, n (%)≥ 15, n (%)
N/A, n (%)
Morphology
Protruding, n (%)
Mixed, n (%)
Nonprotruding and nonexcavated, n (%)
N/A, n (%)
Histology
SLA, n (%)
SHA, n (%)
SCA, n (%)
PGA, n (%)
GCA, n (%)
Small intestinal type, n (%)
Gastric type, n (%)
Adenoma, n (%)
Adenocarcinoma, n (%)
Vienna classiﬁcation
Category 3, n (%)
Category 4, n (%)

Total

CIMP positive

CIMP negative

P value

10664  1091 (86%)15 (14%)25
68  918 (72%)7 (28%)

81
63  1073 (90%)8 (10%)

0.026
0.043

79 (75%)27 (25%)22.9  3.423 (92%)2 (8%)
23.4  3.456 (69%)25 (31%)22.7  3.40.033

29 (27%)77 (73%)4 (16%)
21 (84%)25 (31%)56 (69%)0.20

16 (15%)89 (84%)
1 (1%)
13  861 (57%)42 (40%)3 (3%)

5 (20%)
20 (80%)0 (0%)
17  109 (36%)
14 (56%)2 (8%)

11 (14%)69 (85%)
1 (1%)
12  752 (64%)28 (35%)
1 (1%)

0.52(Bulb versus non-bulb)

16 (15%)5 (5%)
84 (79%)1 (1%)

4 (16%)
0 (0%)
21 (84%)0 (0%)

12 (15%)5 (6%)
63 (78%)1 (1%)

0.78(Protruding or mixed versus nonprotruding)

32 (30%)57 (54%)10 (9%)
2 (2%)
5 (5%)
99 (93%)
7 (7%)
91 (86%)15 (14%)5 (20%)
14 (56%)
3 (12%)
0 (0%)
3 (12%)
22 (88%)
3 (12%)
19 (76%)6 (24%)

27 (34%)43 (53%)
7 (9%)
2 (2%)
2 (2%)
77 (95%)4 (5%)
72 (89%)9 (11%)

0.51*

34 (32%)72 (68%)5 (20%)
20 (80%)29 (36%)52 (64%)

0.14

0.39

0.032
0.032

0.35
0.18

CIMP, CpG island methylator phenotype; GCA, gastric-type intramucosal adenocarcinoma; N/A, not available; PGA, pyloric gland adenoma; SCA, small intestinal-type
intramucosal adenocarcinoma; SHA, small intestinal-type high-grade adenoma; SLA, small intestinal-type low-grade adenoma.
*Analyzed with SLA, SHA, and SCA.

© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
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R Ota, T Sawada et al

was no relationship between CIMP-H status and age, gender, smoking status, or disease location, CIMP-H positivity
was associated with BMI (t-test, p = 0.022) and lesion size
(t-test, p = 0.012) (supplementary material, Table S4). In
addition, the prevalence of CIMP-H tumors did not signiﬁcantly differ among histological subtypes or between small
intestinal-type and gastric-type tumors.

Targeted amplicon sequencing of non-ampullary
duodenal lesions
We performed targeted sequencing of all exons in
75 cancer-related genes frequently mutated in SBAs,
including DAs, as well as duodenal adenomas. A
sequencing overview, including reads, coverage, anduniformity of the read coverage distribution, is shownin supplementary material, Table S5. Each FFPE sample
underwent an average of 8.5 million sequencing reads
after quality ﬁltering. A mean coverage depth of2461.0 reads (737.0–10 950.0) per base was observed.
All single nucleotide variations and insertions/deletions
detected through bioinformatics analysis underwent
visual inspection using the Integrative Genomics Viewer
for conﬁrmation. We identiﬁed a mean of 1.9 somatic
nonsynonymous mutations (range 0–8) per sample (supplementary material, Table S6). At least one somatic
nonsynonymous mutation was observed in 45 of the75 genes. The ten most commonly mutated genes in
non-ampullary duodenal lesions are illustrated in
Figure 2. APC was the most frequently mutated gene inthese lesions (56 of 102 samples; 55%), followed by
KRAS (13/102; 13%), LRP1B (10/102; 10%), GNAS
(8/102; 8%), ERBB3 (7/102; 7%), and RNF43(6/102; 6%).
For APC, a single mutation per sample was detected in
48 subjects, and two different mutations per sample weredetected in eight subjects, resulting in a total of 64 mutations in this study. Most APC mutations were nonsense(43 mutations) or frameshift (16 mutations), though anumber of missense mutations (ﬁve mutations) werealso detected. The mutation distribution within APC
was visualized using MutationMapper in cBioPortal(https://www.cbioportal.org) [40,41] with several modiﬁcations (Figure 3A). Most of the deleterious mutations
were distributed between codons 700 and 1200 or
between codons 1400 and 1600. Although a prior study
suggested that T1556fs is a mutation hotspot within APC
in duodenal adenomas [24], we most frequently found amutation at R1450X (in ten samples), which is similar to
CRC [42] but different from ampullary carcinoma [34](Figure 3B). Although the frequencies of mutations
within mutation cluster regions (codons 700–1200 and
1400–1600) among the total mutations detected in eachhistological type were similar between SLAs and SHAs,
the mutation frequency in small intestinal-type adenomas (SLAs + SHAs) (86%; 51/59 mutations) withinthese regions was higher than in SCAs (33%; 1/3 mutations) (Fisher’s exact test, p = 0.065). Overall, mutations
within WNT signaling pathway components weredetected in 59% (60/102) of the samples (Figure 4).

There were signiﬁcant differences in the prevalence of
KRAS mutations among the histological subtypes ofsmall intestinal-type tumors: SLA (1/32; 3%), SHA(4/58; 7%), and SCA (2/5; 40%) (chi-squared test,
p = 0.013), though there was no signiﬁcant differencein the prevalence of APC mutations among histologicalsubtypes (Table 2). TP53 mutations were detected in ﬁve
samples (5%), and samples harboring a mutation within
at least one gene among APC, KRAS, and TP53 wereobserved in 62 patients (61%) (Figure 2). All eightdetected GNAS mutations were at codon 201, and sixof those are associated with KRAS mutations. When
compared between small intestinal-type tumors andgastric-type tumors, GNAS and KRAS mutations weresigniﬁcantly associated with gastric-type tumors(Table 2).
ERBB2 mutations, which have been frequently
reported in SBAs, were also detected in four of the
102 samples (4%). The frequency of lesions harboringat least one mutation in an ERBB receptor family member (ERBB2, ERBB3, or ERBB4) was 12% (12/102)(Figure 4). BRAF mutation was detected in ﬁve samples(5%), which is comparable to earlier studies reporting
frequencies of 6–11% in SBA samples [17,20,21].
BRAF V600E mutation was detected in only one SCA
sample with CIMP and CIMP-H (Figure 1), which isconsistent with an earlier report that only about 10% of
BRAF-mutated SBAs harbor V600E mutations [20].

Targeted amplicon sequencing detects CNVs
We also detected CNVs in segments of the genome that
could be duplicated or deleted from the sequencing data
(Figure 2 and supplementary material, Table S7). Basedon copy number gains in all samples, the most frequently
affected genes that were considered oncogenic were
EPHA6 (43/102; 42%), followed by KRAS (30/102;29%), ERBB4 (25/102; 25%), BRAF (12/102; 15%),and GNAS (13/102; 13%). On the other hand, the mostfrequently affected tumor suppressive genes by copy
number losses were MIB2 (41/102; 40%), CDKN2A(37/102; 36%), TP53 (10/102; 10%), and ARID1B(10/102; 10%). The distributions of CNVs were not different among tumor histological subtypes. As for ERBB
family genes, copy number gains were observed in one
case each for ERBB2 and ERBB3, while frequent copy
number gains (25/102, 25%) were detected in ERBB4(Figure 4). Lastly, the frequency of lesions harboring atleast one mutation or copy number gain in an ERBB
family member was 34% (35/102).

Immunohistochemistry
Of the 102 non-ampullary duodenal lesions analyzed,
33 (32%) showed focal nuclear expression and
54 (53%) showed diffuse nuclear expression, whereas
15 (15%) showed no nuclear expression of β-catenin(Table 2, Figure 4, and supplementary material,
Figure S2). The prevalence of diffuse and focal nuclear
β-catenin accumulation in duodenal adenomas (SLA,© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 23 -J Pathol 2020; 252: 330–342
www.thejournalofpathology.com

Mutation and methylation in duodenal intramucosal neoplasia

337

Figure 2. Mutation and CNV proﬁles in non-ampullary duodenal adenomas and intramucosal adenocarcinomas. Upper panels show histological types, Vienna classiﬁcation, and CIMP status. Middle panels show summarized results for targeted sequencing of cancer-related genes intumors with the indicated histological types. Lower panels show frequently detected CNVs. Frequencies of CIMP status as well as mutations
and CNVs in respective genes are shown on the right. CIMP, CpG island methylator phenotype; GCA, gastric-type intramucosal adenocarcinoma; PGA, pyloric gland adenoma; SCA, small intestinal-type intramucosal adenocarcinoma; SHA, small intestinal-type high-grade adenoma; SLA, small intestinal-type low-grade adenoma.

SHA, and PGA) (90%; 83/92) was consistent with earlier
reports of nuclear β-catenin accumulation in 64–84% of
non-ampullary adenomas [25–27]. Lesions showing
nuclear β-catenin accumulation (diffuse or focal) were
not associated with APC mutation-positive tumors ortumors with mutations of WNT signaling components.
The prevalence of nuclear β-catenin accumulation was
signiﬁcantly higher in small intestinal-type tumors(86/95, 91%) than in gastric-type tumors (1/7, 14%)
(Fisher’s exact test, p < 0.0001, Table 2). However, when
we focused on tumors with the small intestinal phenotype,the prevalence of nuclear β-catenin accumulation did not
signiﬁcantly differ among SLAs, SHAs, and SCAs. We
found no signiﬁcant differences between β-catenin
expression or molecular variables and the locations (proximal and distal) of the 90 small intestinal-type tumors(supplementary material, Table S3).
Among MMR proteins, expression of MLH1 and MSH2
was evaluated immunohistochemically across all nonampullary duodenal lesions. All samples stained positively

for MLH1, which was largely consistent with the methylation data. By contrast, loss of MSH2 expression was
detected in one SCA sample (KT23) (supplementary material, Figure S3). That sample harbored the largest number ofsomatic mutations (eight) among the 75 genes investigated(supplementary material, Table S6). Six of the eight mutations were insertions of one base resulting in frameshift
mutations, which supports the possibility of MSI.

Discussion
In the present study, we performed integrated genetic
and epigenetic analyses of non-ampullary duodenal
lesions. In addition to clinicopathological variables,
there were signiﬁcant differences in molecular variables between small intestinal-type and gastric-type
tumors. High prevalences of KRAS and GNAS mutations in PGAs and GCAs were consistent with earlier© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 24 -J Pathol 2020; 252: 330–342
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R Ota, T Sawada et al

Figure 3. APC mutations detected in non-ampullary duodenal lesions. (A) Schematic representation of 64 APC mutations detected in the present study. Most of the somatic mutations detected in SLAs and SHAs were clustered within codons 700–1200 and 1400–1600, where hotspot mutation (R1450X) is located. Each circle represents an individual mutation in each patient. (B) Mutations previously reported inampullary carcinomas [34] and colorectal cancers [42] are also indicated for comparison. GCA, gastric-type intramucosal adenocarcinoma;
PGA, pyloric gland adenoma; SCA, small intestinal-type intramucosal adenocarcinoma; SHA, small intestinal-type high-grade adenoma; SLA,
small intestinal-type low-grade adenoma.

reports [43,44]. The low prevalences of nuclear
β-catenin expression in PGAs and GCAs, which isalso consistent with an earlier study [26], may suggestthat the WNT signaling pathway is less involved indevelopment of non-ampullary duodenal tumors withthe gastric phenotype. These results indicate that
small intestinal-type and gastric-type tumors arise
via separate carcinogenic pathways. When small
intestinal-type tumors were divided based on whether
they were located within the proximal or distal duodenum, SLAs and nonprotruding and nonexcavatedtype tumors were more prevalent in the distal part.
However, there were no signiﬁcant differences in
the molecular characteristics including β-catenin

expression between tumors in these two locations.
Previous reports suggested that development ofgastric-type tumors in the proximal duodenum ispotentially associated with gastric acid and H. pylori
infection, whereas small intestinal-type tumors in
the distal duodenum may be associated with bile acids[26]. Further study will be necessary to clarify thepathological and molecular differences between
tumors in the proximal and distal duodenum.
We also found that CIMP and CIMP-H frequencies
are higher in intramucosal adenocarcinomas than in adenomas, indicating that concurrent methylation of CGIs is
likely associated with malignant transformation of nonampullary duodenal adenomas. In the clinical settings,© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 25 -J Pathol 2020; 252: 330–342
www.thejournalofpathology.com

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339

Figure 4. Mutation and copy number gains of genes related to the WNT signaling pathway and ERBB receptor family members in nonampullary duodenal lesions. Pathological types (small intestinal or gastric type) and CIMP status, mutations in WNT signaling-associatedgenes, and β-catenin expression status are shown at the top. Mutations and copy number gains in ERBB receptor family members are shownin the middle panels and lower panels. Frequencies of mutations and copy number gains in respective genes as well as β-catenin expression
positivity are shown on the right. CIMP, CpG island methylator phenotype.

tumors classiﬁed as category 3 according to the revised
Vienna classiﬁcation (SLAs and PGAs) can be monitored and followed up [30], while it is recommended thatcategory 4 tumors (SHAs, SCAs, and GCAs) be treated,
especially those that are 20 mm in diameter or larger, as
these lesions have a high risk of progression to adenocarcinoma [9]. The fact that CIMP or CIMP-H positivity
was associated with larger lesions in the present study
may support this recommendation. At the same time,from the viewpoint of aberrant DNA methylation, SHAs
could be followed up at a later time, especially if they aresmall in size. Despite recent advances in endoscopic
technology, including magnifying endoscopy and image
enhanced endoscopy, it remains difﬁcult to distinguishbetween lesions that should be followed up or treated[6]. Pretreatment diagnosis by using biopsy is also difﬁcult because the biopsy procedure itself may induce
unintended ﬁbrosis, possibly causing unsuccessfulendoscopic resection. If speciﬁc endoscopic ﬁndings
for lesions with concurrent methylation are determined,
detailed endoscopic assessment could contribute to theprediction of premalignant lesions, as was previously
reported for colorectal serrated lesions [45].
MLH1 methylation has been reported in 12% of nonampullary adenomas [16] as well as in 14% [15] or

25% [14] of DAs. MSI has also been reported in 20%[15] or 33% [17] of DAs, where it is reportedly associated with MLH1 methylation [15]. In the present study,
only two MLH1 methylation-positive lesions, with relatively low methylation levels, and no samples with lossof MLH1 immunoreactivity were detected. Additionally, there was only one sample with loss of MSH2
expression in possible association with MSI. These
results suggest that acquisition of MSI in association
with MMR deﬁciency, especially with MLH1 methylation, is an infrequent event in early duodenal
carcinogenesis.
Recent studies indicated that the genes recurrently
mutated in SBAs are TP53 (41–58%), KRAS
(27–54%), and APC (11–27%) [17–22]. An earlier study
analyzed 50 hotspot mutations in cancer-related genes in
19 patients with non-ampullary duodenal adenomas andadenocarcinomas, and found prevalent mutations in
KRAS (63%), APC (47%), and TP53 (37%) [24]. In thepresent study, we found mutations in APC (55%) andKRAS (13%); TP53 mutations were observed in only
5% of the samples. These differences are likely due tothe proportion of the adenocarcinomas or high-grade
adenomas. In the present study, most KRAS mutations
appeared to occur at the progression of intramucosal© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 26 -J Pathol 2020; 252: 330–342
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adenocarcinomas. In addition, the lower prevalence ofTP53 mutations in early duodenal lesions than in SBAs
suggests that most TP53 mutations occur at a later stageof tumorigenesis. These results in SBAs appear to somedegree consistent with the adenoma–carcinoma
sequence previously seen in CRC [10]. We also detecteda higher prevalence of gene mutations associated with
the WNT signaling pathway (59%) and nuclear
β-catenin accumulation (85%) in non-ampullary duodenal lesions. Together with earlier immunohistochemicaland gene expression studies of duodenal adenomas[25–27], these results indicate the importance of the
WNT signaling pathway to duodenal adenoma
development.
Interestingly, we detected APC mutations (55%) more
frequently in early non-ampullary duodenal lesions than
did earlier studies in advanced lesions, which reported
frequencies of 13–27% in SBAs or 8% in DAs[17,20–22]. Kojima et al separately analyzed multiple
components in non-ampullary duodenal lesions thatexhibited different histological grades, and observed
APC mutations more frequently in the adenoma (58%)
than in the adenocarcinoma (25%) components [24].
The earlier observation that abnormal nuclear localization of β-catenin is more frequent in non-ampullary duodenal adenomas than in adenocarcinomas (84% versus
33%) may support these results [26]. Although a numberof studies have reported that gastric intramucosal neoplasias with the small intestinal phenotype also frequently show APC mutations, a few dysplasia/
intramucosal neoplasias with APC mutation reportedly
progress to gastric cancer [46]. Together with the different mutation distribution patterns between duodenal
adenomas (SLAs + SHAs) and SCAs, the higher prevalence of APC mutations in duodenal adenomas than in
SBAs or DAs may suggest that most duodenal adenomas, especially those with mutations within mutation
cluster regions, also have low malignant potential anddo not progress to DA. This suggests that the
adenoma–carcinoma sequence has only limited impact
on duodenal carcinogenesis. Targeted deep DNA
sequencing with tumor variant allele frequency analysis
in larger numbers of adenoma, intramucosal adenocarcinoma, and DA samples may conﬁrm these ﬁndings andimprove our understanding of the mechanisms underlying duodenal carcinogenesis, as has been described forgastric intramucosal neoplasias with the small intestinal
phenotype [46].
ERBB2 mutations and ampliﬁcations have been
detected in 12–23% of SBAs, which suggests that they
are potential therapeutic targets [17,20–22]. ERBB2
alterations are also signiﬁcantly associated with a duodenal location when compared to other parts of the small
intestine [17,20]. In the present study, ERBB2 mutations
were observed in only 4% of samples. Because a significant number of ERBB2 mutations were observed in
SBAs with MMR deﬁciency [17,21], most of thesemutations may have been acquired after the cancer progression and/or acquisition of MSI. A recent study using
SBA patient-derived cell lines demonstrated that small-molecule ERBB2 inhibitors have anti-cancer activity
both in vitro and in vivo [22]. Such small-molecule
ERBB2 inhibitors may be useful not only for the treatment of metastatic SBA but also for the chemoprevention of multiple non-ampullary duodenal adenoma inpatients with FAP, if activating ERBB2 mutations aredetected.
As for CNVs, previous conventional and array comparative genomic hybridization studies reported that in
SBAs, the regions most commonly exhibiting copy
number gains were at chromosomes 5p, 7, 8q, 13, 16,
and 20, while copy number losses were detected at chromosomes 4, 5q, 8p, 17p, and 18 [23]. In the present
study, the frequent occurrence of copy number losses
at the TP53 locus (17p) as well as the gains at the BRAF
(7q) and GNAS (20q) loci was consistent with those earlier results. Notably, frequent copy number gains at the
ERBB4 locus were observed in the present study. In
ovarian serous carcinoma, immunohistochemical detection of high ERBB4 expression reportedly correlatedwith cisplatin resistance and a poorer prognosis [47].
Because recent studies suggest that somatic CNVs at
oncogenic loci are not always associated with gene
expression [42,48,49], further validation of the effectof CNVs through comparison with expression is needed
before the utilization of CNVs as biomarkers.
This study has several limitations. One is the lack of
MSI analysis other than the immunohistochemical comparison of MMR proteins. In addition, the sample sizes
were small when the specimens were divided into
intestinal- and gastric-type tumors or CIMP-H andnon-CIMP-H tumors. Furthermore, analysis of corresponding normal samples for validation of somatic
mutations was not performed. Although rigorous bioinformatics pipelines were used to discriminate somaticfrom germline mutations, the results of mutation analyses may still include germline mutations. However,
we have several novel ﬁndings. First, molecular andclinicopathological characteristics differ among small
intestinal-type tumors and gastric-type tumors, which issuggestive of separate carcinogenic pathways. Second,
concurrent methylation of CGIs appears to be associatedwith the development of non-ampullary duodenal
lesions. Third, prevalent WNT pathway gene mutations
and positive staining for nuclear β-catenin indicate theinvolvement of WNT signaling in the development of
duodenal tumors. Finally, we observed a higher frequency of APC mutations than previously reported in
SBA patients as well as differences in the mutation distributions within APC between adenoma and intramucosal adenocarcinoma samples. This suggests that the
adenoma–carcinoma sequence has only limited involvement in duodenal carcinogenesis.
At present, it remains difﬁcult to predict the progression from adenoma to adenocarcinoma or to determine
endoscopically which lesions can be followed and whichmust be treated. The differences between the pathogenesis of small intestinal-type tumors in the proximal duodenum and those in the distal part are alsouncharacterized. Accumulation of data from© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 27 -J Pathol 2020; 252: 330–342
www.thejournalofpathology.com

Mutation and methylation in duodenal intramucosal neoplasia

comprehensive genetic and epigenetic analyses andcomparison with more detailed endoscopic and pathological ﬁndings will likely provide new insight into
non-duodenal ampullary carcinogenesis as well as theendoscopic diagnosis of early non-ampullary duodenal
lesions.

Acknowledgements
We thank Mutsumi Toyota, Saeko Ishioka, Isao
Kurahayashi, Noriko Sasahara, Satomi Saito, Atsuko
Asaka, Naoko Abe, and Aki Iwata for their excellent
technical assistance; Rie Oka for helpful advice for statistical analyses; and Dr William F Goldman for editing
the manuscript. This study was supported in part by
JSPS KAKENHI Grant Nos JP16K09304 andJP19K08463 (to TS), JP17K09374 (to SI), JP19K08367(to RO), and 16H06279 (PAGS) (to TT); a Grant-inAid from the Takeda Science Foundation (to TS); andan Extramural Collaborative Research Grant of the Cancer Research Institute, Kanazawa University (to YS).

Author contributions statement
RO and TS proposed the concept, contributed to thestudy design, performed statistical analysis, and wrote
the manuscript. RO, TS, ST, YK, HM, KN, SK, TY
and TM contributed to the collection and interpretationof pathological and immunohistochemical data. RO,
TS, YS, HS, EY, EK, HK and TT contributed to the collection and interpretation of molecular data. RO, KH,
HN, SI, ST, NY and HD contributed to the collectionof clinical data. ST, YS, HS, TY and TM assisted withdraft revision of the manuscript. All the authors
approved the ﬁnal draft submitted for publication.

Data availability statement
Access to the data from our study is available upon reasonable request.

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SUPPLEMENTARY MATERIAL ONLINE
Figure S1. Representative views of duodenal adenomas and intramucosal adenocarcinomas (H&E staining)
Figure S2. Representative views of H&E staining (left) and β-catenin nuclear expression (right)
Figure S3. Representative views of immunohistochemistry analysis of mismatch repair (MMR) proteins
Table S1. Primer sequences used in this study
Table S2. List of genes in the custom Ampliseq gene panel used in this study
Table S3. Comparison of clinicopathological and molecular characteristics between small intestinal-type non-ampullary duodenal lesions located in theproximal and distal parts of the duodenum
Table S4. Relationship between CIMP-high status and clinicopathological characteristics of non-ampullary duodenal lesions
Table S5. Summary of targeted amplicon sequencing data
Table S6. Somatic nonsynonymous mutations found in 102 non-ampullary duodenal lesions
Table S7. Copy number alterations found in 102 non-ampullary duodenal lesions© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd.
on behalf of The Pathological Society of Great Britain and Ireland. www.pathsoc.org- 29 -J Pathol 2020; 252: 330–342
www.thejournalofpathology.com

MOLECULAR AND CLINICAL ONCOLOGY 15: 235, 2021

Thymidylate synthase locus LOH in combination with genotype
has prognostic and predictive significance in colorectal cancer
MASANORI KOTAKE1‑4, HIROYUKI BANDO2, MAMI KANEKO1,2,4, HIROFUMI TAKEMURA1,TOSHINARI MINAMOTO4 and KAZUYUKI KAWAKAMI4,5
1

Department of General and Cardiothoracic Surgery, Graduate School of Medical Science, Kanazawa University,
Kanazawa, Ishikawa 920‑8641; 2Department of Gastrointestinal Surgery, Ishikawa Prefectural Central Hospital,
Kanazawa, Ishikawa 920‑8530; 3Department of Surgery, Kouseiren Takaoka Hospital, Takaoka, Toyama 933‑8555;
4
Division of Translational and Clinical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa,
Ishikawa 920‑0934; 5Department of Chemotherapy and Palliative Care, Tokyo Women's Medical University,
Tokyo 162‑8666, Japan
Received September 29, 2020; Accepted July 22, 2021
DOI: 10.3892/mco.2021.2398
Abstract. The aim of the current study was to investigate theprognostic and predictive significance of polymorphisms in thethymidylate synthase (TS) gene, alongside the loss of hetero‑
zygocity (LOH) at this gene locus in patients with colorectal
cancer. Genotyping was carried out for a variable number
tandem repeat (VNTR) polymorphism in the TS 5'‑untranslated
region, a G/C single nucleotide polymorphism (SNP) locatedwithin this VNTR, and for TS LOH status in 246 colorectal
cancer and paired normal DNA samples. The results wereanalyzed in relation to clinicopathological features, including
the prognostic and predictive significance of TS genotype inpatients who underwent curative surgery. Complete VNTR,
SNP and LOH information for TS was obtained in 226 cases.
No significant associations were observed between normal
tissue TS genotype status and clinicopathological features.
LOH of TS was observed in 58% of tumor samples and was
associated with poor prognosis independently of clinical stage.

Correspondence to: Dr Masanori Kotake, Department ofSurgery, Kouseiren Takaoka Hospital, 5‑10 Eiraku‑cho, Takaoka,
Toyama 933‑8555, JapanE‑mail: kotake628@gmail.com
Professor Toshinari Minamoto, Division of Translational andClinical Oncology, Cancer Research Institute, Kanazawa University,
13‑1 Takara‑machi, Kanazawa, Ishikawa 920‑0934, JapanE‑mail: minamoto@staff.kanazawa‑u.ac.jp

Abbreviations: CRC, colorectal cancer; LOH, loss ofheterozygosity; PE, primer extension; RFLP, restriction fragment
length polymorphism; SNP, single nucleotide polymorphism;
TS, thymidylate synthase; 5'‑UTR, 5'‑untranslated region;
VNTR, variable number tandem repeatKeywords: 5‑fluorouracil, colorectal cancer, genetic
polymorphism, loss of heterozygocity, thymidylate synthase

Cases exhibiting TS LOH were classified into the three groupsof 2R/loss, 3G/loss and 3C/loss. Patients with 3C/loss genotype
status had poor outcomes when treated by surgery alone, but
their survival was similar to patients with other genotypes
following Fluorouracil (5‑FU)‑based adjuvant chemotherapy.
The results suggested that LOH of the TS locus may be asignificant prognostic factor in colorectal cancer, with the
genotype of the residual allele also demonstrating an influenceon prognosis. In conclusion, LOH status should be consideredwhen TS genotype is explored as a potential prognostic andpredictive marker for 5‑FU‑based adjuvant chemotherapy incolorectal cancer.
Introdution
Colorectal cancer (CRC) is one of the most common
malignancies worldwide. Although most localized cases of thedisease are treated surgically, a considerable number of patients
experience disease recurrence. Adjuvant chemotherapy after
curative surgery has been shown to reduce the recurrence rate
and therefore all CRC patients with clinical stage III disease
are recommended to receive adjuvant chemotherapy (1), eventhough only a proportion of these derive a benefit. Patients withclinical stage II disease are mostly treated with surgery alone,
however some may benefit from adjuvant therapy because of ahigh risk of recurrence (2,3). To maximize the efficacy of adju‑
vant chemotherapy, accurate predictive markers are needed to
select patients who will benefit most from treatment.
5‑FU‑based chemotherapy is the current standard of care
for adjuvant therapy following surgical treatment of CRC (4).
Thymidylate synthase (TS) is a target enzyme for 5‑FU (5),leading to extensive studies of TS mRNA expression (6), TS
protein expression (7,8) and TS gene polymorphisms (9,10)
as potential predictive factors for the efficacy of 5‑FU‑based
adjuvant chemotherapy. Currently however, no informa‑tion regarding TS status is recommended for routine
clinical use in the selection of patients to receive 5‑FU‑based
chemotherapy (11).

- 30 -

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KOTAKE et al: LOH OF TS HAS PROGNOSTIC AND PREDICTIVE SIGNIFICANCE IN CRC

TS shows unique genetic variants comprising a vari‑able number of tandem repeat (VNTR) and a single
nucleotide polymorphism (SNP) in its 5' untranslated region(5'UTR) (12‑14). These may be predictive markers for 5‑FU
efficacy and for adverse events from this treatment. We previ‑
ously reported that VNTR and SNP can give rise to four TS
allele types: 2G, 2C, 3G and 3C. These may affect the trans‑
lational activity of TS mRNA, thus influencing TS protein
expression and therefore constitute a marker for the efficacyof 5‑FU‑based adjuvant chemotherapy (14‑16). In addition tothese four allele types, other rarer alleles comprising morethan three repeats and novel SNPs in the 2R allele have also
been reported (17), thus giving rise to a larger number ofallele types. Furthermore, we observed that frequent loss ofheterozygosity (LOH) of the TS locus in tumors can affect
the genotype, thereby indirectly influencing the TS expres‑sion level in tumors (18,19). This potential change in genotype
status due to LOH should be considered in studies of the TS
genotype as a predictive marker. The status of VNTR, SNPs
in both 2R and 3R, and LOH must all be evaluated before
TS genotype information can be introduced into the clinicalsetting.
In this study, we analyzed the TS VNTR, the SNPs in boththe 2R and 3R alleles, as well as the LOH status of the TS locus
in order to explore their potential significance as prognostic
and predictive markers of 5‑FU‑based adjuvant chemotherapy
in CRC.
Materials and methods
Patient cohort and DNA isolation. Matched tumor and normal
tissue samples were obtained following surgical resection
for primary colorectal adenocarcinoma in 246 patients. The
patients were all Japanese and comprised 146 males and
100 females, ranging in age from 33 to 93 years (mean age
66.0 years). The resected tissues were fixed in formalin andembedded in paraffin followed by H&E staining and histo‑
logical diagnosis. Tumor tissue was dissected manually from
10 µm sections of formalin‑fixed, paraffin‑embeded tissue
blocks. After deparaffinization using xylene and ethanol,
genomic DNA was isolated using a QIAamp DNA FFPE
Tissue Kit (QIAGEN, Hilden, Germany) following the protocol
provided by the manufacturer. Approval for this project
was obtained from the Kanazawa University Genome/GeneAnalysis Research Ethics Committee.
Genotyping of TS VNTR and SNP. TS genotypes for the
VNTR and the SNP in the 3R allele were determined by
PCR and PCR‑restriction fragment length polymorphism(RFLP) using the forward primer TS25: 5'‑AGG CGC GCG
GAAGGG GTCCT‑3' and reverse primer TS18: 5'‑TCCGAG
CCGGCCACAGGCAT‑3' as described previously (14) with amodification of PCR conditions. PCR with the genomic DNA
template was performed in reaction mixtures containing 1X
TaKaRa HS Taq buffer (TaKaRa Bio, Otsu, Japan), 200 µM
deoxyribonucleoside triphosphates, 500 nM of each primer,
0.5 unit of TaKaRa HS Taq DNA polymerase (TaKaRa Bio)and 100 ng of genomic DNA. The cycling conditions were: one
cycle at 95˚C for 3 min, 35 cycles at 98˚C for 10 sec and 68˚C
for 60 sec, with a final extension at 72˚C for 5 min. Aliquotsof amplified fragments were separated on 3% agarose gels todetermine the TS VNTR genotype.
Samples showing the 2R/3R or 3R/3R genotypes wereanalyzed further for the G/C polymorphism in the 3R allele
by using the RFLP method. HaeIII digestion of the 3R frag‑ment produced 66‑, 37‑, 28‑ and 10‑bp bands for the 3G allele,
and 94‑, 37‑ and 10‑bp bands for the 3C allele after separation
on 3% agarose gels. For samples with 2R/2R or 2R/3R geno‑
types, the G/C polymorphism in the 2R allele was determinedby PCR‑PERFLP method, consisting of PCR followed byprimer extension (PE) and RFLP analysis with HaeIII diges‑tion. The PCR reaction was performed using reverse primer
TS21: 5'‑CAGCTCCGAGCCGGCCACAG‑3' instead of TS18.
Five microliters of PCR product was mixed with extension
primer TS105: 5'‑TCCGAGCCAGCCACAGGCAT‑3' labeledwith fluorescein 5'‑isothiocyanate to a total volume of 7.5 µl.
The mixture was denatured for 5 min at 98˚C, annealed for
10 min at room temperature and then combined with 2.5 µlof PE reaction mixture containing 0.5 unit of Vent (exo‑)
DNA polymerase (New England BioLabs, Ipswich, MA),
200 µM deoxyribonucleoside triphosphates, 1X ThermoPol
Reaction Buffer provided by the manufacturer, followed byincubation for 10 min at 72˚C. The product of primer exten‑
sion was digested with HaeIII, separated on 3% agarose gelsand visualized with the Typhoon fluoroimager. The 2G allele
produced a 48 bp fragment and the 2C allele a 76 bp fragmentwith the fluorescein 5'‑isothiocyanate label. The TS genotype
was thus classified into 2G/2G, 2G/2C, 2C/2C, 2G/3G, 2G/3C,
2C/3G, 2C/3C, 3G/3G, 3G/3C, or 3C/3C by comprehensive
genotyping of the VNTR and SNP in the TS 5'UTR. Analyses
were performed at least twice to confirm the genotype.
LOH analysis. LOH of the TS locus was determined in three
distinct ways depending on the TS genotype observed in thenormal tissue. The G/C SNP in the 2R allele was not taken
into consideration for LOH analyses. Samples that were
2R/3G or 2R/3C were analyzed by PCR followed by separa‑tion on Spreadex gel (Elchrom Scientific, Cham, Switzerland).
Samples that were 2R/2R, 3G/3G or 3C/3C were evaluated for
LOH using the microsatellite marker D18S59, as described
previously (18). Samples that were 3G/3C were analyzed using
the PCR‑PERFLP method, as described above for the SNP
genotyping method with the 2R allele. PCR‑PERFLP avoids
interference due to heteroduplex formation, thereby allowing
the exact allele ratio to be determined. The 3G allele produced
a 76 bp fragment and the 3C allele a 104 bp fragment with
PCR‑PERFLP, as visualized by Typhoon fluoroimaging. The
image was analyzed using ImageQuant software and therelative ratio between 3G and 3C alleles in tumor DNA wasnormalized using the ratio measured in the corresponding
normal tissue DNA sample. LOH was defined as either thecomplete absence of one allele, or a decrease in intensity of
one allele by at least 50%. LOH of 18q was analyzed using the
microsatellite markers D18S58, D18S61 and D18S64. Forward
primers were labeled with fluorescein 5'‑isothiocyanate and thesame method as for microsatellite marker D18S59 was used.
Statistical analysis. Relationships between variables wereanalyzed by Chi‑square analysis or the Scheffe post‑hoc test
used following ANOVA. The cumulative survival rate was- 31 -

MOLECULAR AND CLINICAL ONCOLOGY 15: 235, 2021

3

estimated using the Kaplan‑Meier method and statisticalsignificance was assessed by the log‑rank test. Cox regression
modelling was used for multivariate analysis. P‑values lessthan 0.05 were considered significant.
Results
Genotype analysis in normal tissue samples. We previously
investigated and compared the TS genotypes between matchednormal and tumor tissues from 151 patients with colorectal
cancer. The results suggest that frequent LOH of the TS locus
detected in the tumors affect the functional TS genotyping (18).
Therefore, in the present study, TS genotyping was carried out
on DNA from normal tissue rather than from tumor samples
in order to avoid possible artifacts from LOH. The VNTR
genotype distribution amongst the 246 cases was: 2R/2R (n=9),2R/3R (n=70), 3R/3R (n=162) and 3R/5R (n=5). Because the
5R allele is rare and analysis of the G/C SNP in the repeat
component is difficult, the 5 cases with 3R/5R genotype were
excluded. The remaining 241 cases of 2R/2R, 2R/3R and 3R/3R
VNTR genotype cases were then screened for the G/C SNP
in both the 2R and 3R alleles (Fig. 1). The combined VNTR
and SNP genotype frequencies were: 2G/2C (n=1), 2C/2C(n=8), 2G/3G (n=3), 2G/3C (n=3), 2C/3G (n=25), 2C/3C (n=39),
3G/3G (n=48), 3G/3C (n=81) and 3C/3C (n=33). The previ‑
ously reported 2R allele with a G→C SNP located in the first
tandem repeat (17) was not found in our subjects. This shouldhave resulted in a 121 bp fragment following PCR‑PERFLP
and gel electrophoresis (Fig. 1B). The 2G allele was quite rarein our population and showed no significant associations withany clinicopathological variable or with clinical course (datanot shown). Therefore, SNP information for the 2R allele was
not considered in further analysis. The distribution of clinico‑pathological features according to TS VNTR/SNP genotype
are shown in Table I. As reported previously (14), the 3G allele
was less frequent in females (P=0.04, chi‑square test) (maleG=115, female G=62, male C=79, female C=68). No significant
associations were apparent between the normal tissue TS geno‑
type and any other clinicopathological feature.
LOH and the residual TS allele in colorectal cancer. The
appropriate method for LOH analysis was selected according
to the genotype found in the normal tissue, as shown in
Fig. 2. The genotype frequency according to LOH status isalso shown in Fig. 2. A novel method for LOH analysis of the
3G/3C genotype was employed in this study and involved theuse of PCR‑PERFLP to avoid interference with heteroduplex
product (Fig. 3). Fifteen cases could not be evaluated for
LOH status because both the TS genotype and the D18S59
microsatellite marker were homozygous. Therefore, complete
TS VNTR/SNP genotype information together with tumor
LOH status was available for 226 patients. The frequencies
were: 2R/2R (n=3), 2R/3G (n=11), 2R/3C (n=20), 3G/3G(n=13), 3G/3C (n=34), 3C/3C (n=14), 2R/loss (n=26), 3G/loss(n=58) and 3C/loss (n=47). The overall frequency of LOH for
the TS locus was 58.0% (131/226). Lossed alleles were evenly
distributed between 2R (n=23), 3G (n=58) and 3C (n=50).
Associations between LOH status and clinicopathologicalfeatures are shown in Table II. The absence of LOH for TS
was significantly associated with proximal tumor location

Figure 1. TS VNTR and SNP analysis. (A) VNTR analysis using PCR ampli‑
fication and separation of products on a 3% agarose gel. (B) SNP analysis in
the 2R allele using PERFLP followed by separation on a 3% agarose gel andscanning with a fluoroimager. (C) SNP analysis in the 3R allele by RFLP and
separation on a 3% agarose gel. The DNA fragments stained using ethidium
bromide are displayed as white pixels on a black background, whereas thoselabeled by fluoresein are displayed as black pixels on a white background.
The numbers on these panels indicate the same samples being analyzed.
The genotypes are as follows: 1, 2G/2C; 2, 2C/2C; 3, 2G/3C; 4, 2C/3G;
5, 2C/3C; 6, 3G/3G; 7, 3G/3C; and 8, 3C/3C. M indicates the size marker(50, 100, 200 and 300 bp) and NC indicates that there was no templatecontrol. TS, thymidylate synthase gene; VNTR, variable number tandem
repeat; SNP, single nucleotide polymorphisml RFLP, restriction fragment
length polymorphism.

and with mucinous histology. No other statistically significant
associations were observed.
Patient prognosis and TS LOH status. A number of studies
have reported that LOH at a given gene locus is associated withpoor prognosis. We therefore analyzed the prognostic signifi‑
cance of TS LOH status before examining the prognostic roleof the TS genotype. This was performed for 153 patients withclinical stage II or III disease who underwent curative surgery
and where clinical information including long term follow‑up
and use of adjuvant therapy was available. Of these patients, 90(59%) showed TS LOH and had significantly shorter survival(P=0.0005) compared to patients with no LOH (n=63; Fig. 4).
We also compared the frequency of LOH between the tumors
at clinical stage II and III by Chi‑square test. We found nostatistical difference in frequency of LOH between the twogroups of tumors (P=0.22). Multivariate analysis with theparameters listed in Table III demonstrated that TS LOH
status, receipt of adjuvant chemotherapy and clinical stage
were independent prognostic factors in this patient cohort.
TS LOH is often accompanied by 18q LOH. TS is located on
18p11.32. Earlier studies reported that 18q LOH was associatedwith poor prognosis in CRC (20,21). Thus, we analyzed the rela‑tion between TS LOH and 18q LOH in 41 randomly selected
samples comprising 15 cases with no TS LOH and 26 caseswith TS LOH. Three microsatellite markers (D18S58, D18S61,
D18S64) located at 18q22.3, 18q22.2 and 18q21.32, respectively,
were used to determine 18q LOH. Fig. 5 shows the TS LOH- 32 -

4

KOTAKE et al: LOH OF TS HAS PROGNOSTIC AND PREDICTIVE SIGNIFICANCE IN CRC

Table I. Thymidylate synthase genotype and clinicopathological features.

Parameter
Total
Sex
Male
FemaleAge (years)
Mean
SD
Stage
I
II
III
IV
Tumor site
Proximal
DistalPathology
Tub1
Tub2
Muc
Por

2R/3R
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
2R/3R
2R/3G
2R/3C

3R/3R
‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
3G/3G
3G/3C
3C/3C

9

28

42

48

81

33

5
4

18
10

23
19

37
11

41
40

19
14

63.3
15.8

70.4
11.1

63.7
11.2

66.3
13.2

65.5
11.1

66.3
13.5

1
5
3
0

4
5
13
6

6
17
17
2

3
18
14
13

7
32
27
15

3
10
13
7

2
7

9
19

12
30

19
29

32
49

9
24

7
1
1
0

8
17
0
3

17
21
1
3

21
23
2
2

39
36
1
5

13
16
2
2

Muc, mucinous adenocarcinoma; Por, poorly differentiated adenocarcinoma; Tub1, well differentiated tubular adenocarcinoma; Tub2,
moderately differentiated tubular adenocarcinoma.

Figure 3. Representative cases of LOH analysis in 3G/3C genotype samples.
The PCR‑primer extention restriction fragment length polymorphism method
detailed in the materials and methods was used to avoid interference by theheteroduplex product. The image of DNA fragments labeled by fluoresein
are displayed as black pixels on a white background. Thymidylate synthasegenotype and LOH status is indicated on the top of matched T and N lanes.
LOH, loss of heterozygocity; T, tumor; N, normal.

Figure 2. Flow chart of LOH analysis and the frequency of TS genotypes.
The LOH status of tumors was analyzed according to the TS genotype innormal sample. The TS genotype frequency in relation to LOH status isshown in the box. LOH, loss of heterozygocity; TS, thymidylate synthasegene; RFLP, restriction fragment length polymorphism.

status as well as that of each 18q microsatellite marker. Whenever
LOH was observed at the TS gene locus, it was also consistently
present at other 18q loci. On the other hand, three tumors (frompatients no. 4, 13 and 16 shown in Fig. 5) showed LOH at one ormore 18q microsatellite markers in the absence of LOH at TS.
In these tumors, LOH was not observed for all three markers,
indicating the chromosomal loss occurred in a relatively small
area of 18q. These results suggest that LOH at TS and 18q are
simultaneous events in most CRC, although a few tumors havesmall areas of LOH at 18q without allelic loss at the TS locus.
TS genotype as a prognostic and predictive factor in tumors
with LOH. Since TS LOH was a strong prognostic factor (Fig. 4)- 33 -

MOLECULAR AND CLINICAL ONCOLOGY 15: 235, 2021

Table II. Loss of heterozygosity of the thymidylate synthaselocus and clinicopathological features.
Parameter

No LOH

LOH

Total
Sex
Male
FemaleAge (years)
Mean
SD
Stage
I
II
III
IV
Tumor site
Proximal
DistalPathology
Tub1
Tub2
Muc
Por

95

131

55
40

80
51

64.6
12.9

66.9
11.3

11
30
39
15

12
52
46
21

48
47

30
101

42
39
6
8

55
69
1
6

P‑value
0.63
0.16
0.61

<0.0001
0.038

5Table III. Multivariate analysis of prognostic factors.
Variable
TS LOHYes vs. no
Adjuvant chemotherapy
Yes vs. no
Sex
Male vs. female
Age≥66 vs. <66
Stage
II vs. III
Tumor site
Proximal vs. distal
Pathology
Tub2 vs. Tub1
Muc vs. Tub1
Por vs. Tub1

Hazard ratio

95% CI

P‑value

3.01

1.36‑6.64

0.00650.53

0.28‑0.98

0.045

1.22

0.66‑2.26

0.53

1.15

0.62‑2.16

0.66

0.53

0.29‑0.96

0.039

0.63

0.28‑1.43

0.27

1.27
2.42
0.53

0.368‑2.38
0.50‑11.7
0.068‑4.21

0.45
0.27
0.55

CI, confidence interval; LOH, loss of heterozygosity; M, mucinous
adenocarcinoma; Por, poorly differentiated adenocarcinoma; TS,thymidylate synthase; Tub1, well differentiated tubular adenocarci‑noma; Tub2, moderately differentiated tubular adenocarcinoma.

Muc, mucinous adenocarcinoma; Por, poorly differentiated adeno‑carcinoma; SD, standard deviation; Tub1, well differentiated tubular
adenocarcinoma; Tub2, moderately differentiated tubular adenocar‑
cinoma.

Figure 4. Overall survival of patients with curatively resected colorectal
cancer according to their TS LOH status. TS, thymidylate synthase gene;
LOH, loss of heterozygocity.

and also influences the TS genotype observed in tumors, we
explored the role of TS genotype separately in patient groups
stratified according to their LOH status. In patients without
TS LOH (n=63), the tumor genotype is identical to that foundin normal tissue. These patients were classified into 6 groups:
2R/2R (n=2), 2R/3G (n=6), 2R/3C (n=13), 3G/3G (n=10),
3G/3C (n=23) and 3C/3C (n=9). The relatively small numberof cases for each genotype prevented analysis of the prognostic
value of these groups. When the genotypes were grouped into
L‑type (2R/2R, 2R/3C, 3C/3C; n=24) and H‑type (2R/3G,
3G/3C, 3G/3G; n=39) according to criteria from our previous
report (14), no prognostic significance was observed in theoverall patient group, in patients treated by surgery alone, or inpatients treated with adjuvant chemotherapy (data not shown).
In patients with TS LOH (n=90), the tumor TS genotypes
were: 2R/loss (n=20), 3G/loss (n=39) and 3C/loss (n=31). No
prognostic significance was observed for these genotypes in theoverall group of patients with TS LOH (Fig. 6A) or in patients
who received adjuvant chemotherapy (Fig. 6C). However, the
3C/loss genotype was associated with significantly shorter
survival in patients treated by surgery alone (Fig. 6B). These
results suggest that patients with the 3C/loss genotype havepoor prognosis when treated by surgery alone, but may benefit
from chemotherapy as observed by the similar survival rate
to patients with other genotypes. Indeed, patients with the
3C/loss genotype who received adjuvant chemotherapy
survived significantly longer than those treated by surgery
alone (Fig. 6D). Thus, the 3C/loss genotype appears to be a
prognostic marker for poor outcome, as well as a predictive
marker for good response to 5‑FU‑based chemotherapy.
Discussion
In this report we genotyped TS for VNTR status and for SNPs
located within the 2R and 3R alleles. We have evaluated the
TS locus for LOH in CRC. In agreement with our previous
observations, LOH was quite frequent regardless of the TS- 34 -

6

KOTAKE et al: LOH OF TS HAS PROGNOSTIC AND PREDICTIVE SIGNIFICANCE IN CRC

Figure 5. LOH status of the TS locus and of 18q in 41 randomly selected tumor samples. White squares indicate that the LOH status could not be determined
either due to microsatellite instability (I) or homozygosity (H). Light grey squares indicate no LOH and dark grey squares indicate LOH. LOH, loss ofheterozygocity; TS, thymidylate synthase gene.

Figure 6. Survival analysis of patients with colorectal cancer exhibiting
TS LOH. The survival rates of patients with 2R/loss (broken line), 3G/loss(dotted line) and 3C/loss (continuous line) genotype were compared using the
Kaplan‑Meier method in (A) all patients with TS LOH, (B) patients treatedwith surgery alone and (C) patients who received adjuvant chemotherapy.
(D) Survival of patients with the 3C/loss genotype who did (broken line) or did
not (continous line) receive 5‑FU‑based adjuvant chemotherapy. TS, thymi‑
dylate synthase gene; LOH, loss of heterozygocity; 5‑FU, Fluorouracil.

genotype (18,19). TS LOH was a prognostic factor for poor
survival (Fig. 4), independently of clinical stage and otherclinicopathological features. Because of the high frequency of
TS LOH (58%) and also its significant prognostic impact, the TS
genotype cannot be combined with LOH status to give one simple
prognostic indicator similar for example to the ‘3G‑containing
type’ we used previously (14). The 3G/3G and 3G/loss genotypes
are identical in that both have only the 3G allele, however their
prognostic significance is quite different due to the presence of
LOH in the latter. Important prognostic information derived
from the LOH status would be lost if the 3G/3G and 3G/loss
genotypes were combined into a simple ‘3G type’.
In exploring the predictive value of a given factor for adju‑
vant chemotherapy, it is also important to consider its prognostic
value in the absence of such treatment. The TS LOH status isessential for the correct use of TS genotype as a prognostic andpredictive factor in 5‑FU‑based adjuvant chemotherapy. The
different number of TS genotypes in tumor DNA is anotherreason to stratify patients according to their TS LOH status.
In cases with no LOH, 6 major TS genotypes are observed(2R/2R, 2R/3G, 2R/3C, 3G/3G, 3G/3C, 3C/3C) whereas threegroups are seen in cases with LOH (2R/loss, 3G/loss, 3C/loss).
Future investigations into the role of TS genotype in the clinicalsetting will require large patient cohorts so that the LOH statusof TS can also be taken into account.
In the current study we classified patients according totheir TS LOH status and then followed by investigating the
prognostic and predictive significance of TS genotype. In caseswith no LOH, the overall patient group showed relatively good
prognosis (Fig. 4) and there were 6 major genotype groups,
making it difficult to obtain statistically meaningful results
because of the relatively small patient numbers. Moreover, noprognostic or predictive significance was observed when these6 genotypes were classified into just two groups (H and L)
according to our previous results (14). This indicates that TS
genotype is not a useful marker in patients without TS LOH,
although study of a larger number of patients is required toconfirm this observation.
Cases with TS LOH showed poor prognosis (Fig. 4). These
were further classified into three simple TS genotype groups(2R/loss, 3G/loss, 3C/loss) in order to explore their prognostic
and predictive values (Fig. 6). The 3C/loss genotype was amarker for poor prognosis in patients treated by surgery alone(Fig. 6B). Furthermore, the 3C/loss genotype also predicted
good response to 5‑FU‑based adjuvant chemotherapy (Fig. 6D).
Despite the relatively small number of patients (n=31), thisresult reached a high level of statistical significance (P= 0.0001).
Using an in vitro reporter assay, we previously showed the 3C
allele was associated with lower translational activity compared- 35 -

MOLECULAR AND CLINICAL ONCOLOGY 15: 235, 2021

to the 3G allele (14). Low expression of TS mRNA (6) andof the TS protein (8) in CRC have both been associated withgood response to 5‑FU‑based chemotherapy. The current result
showing the TS 3C/loss genotype is a marker for good response
to 5‑FU‑based adjuvant chemotherapy (Fig. 6D) is therefore
consistent with our previous in vitro observations and with theresults of Salonga et al (6) and Soong et al (8). Although wecannot explain why this genotype was associated with poor
prognosis (Fig. 6B), the result concurs with a previous study
showing that low TS expression is a marker of worse prognosis
in patients treated by surgery alone (8).
Due to the retrospective nature of this study and the poten‑
tial for biases, further analyses are required to validate theresults, particularly for the TS genotype groups in cases with
LOH. The 2G allele was rare and no 2R allele with the G→C
SNP in the first tandem repeat was found in our patient cohort,suggesting that SNP typing of the 2R allele can be omitted
in further studies of the Japanese population. However, there
is considerable ethnic variation in the allele frequency for
2R and the incidence is higher in Western populations (22).
Therefore, additional analysis of the SNP in the 2R allele may
be required for Caucasian populations.
The LOH status of TS was closely associated with 18q
LOH status, with the latter being reported previously as aprognostic factor in CRC (23,24). The mechanism by which
18q LOH is linked to poor prognosis of CRC patients is notknown, although the loss of several tumor suppressor genes
in this region including DCC, SMAD4 and SMAD22 has been
implicated. The current study sheds light on loss of the wholeof chromosome 18 as a prognostic factor. LOH of 18p and
18q should be analyzed simultaneously to investigate whether
the minimally lost regions on 18q or the whole allelic loss ofchromosome 18 have stronger prognostic significance.
TS LOH is a significant prognostic factor in CRC.
Furthermore, the 3C/loss genotype appears to be prognostic
in patients treated by surgery alone and predictive in patients
who receive 5‑FU‑based adjuvant chemotherapy. Since TS
LOH status influences the TS genotype of tumors and also
has a significant prognostic role, TS LOH should be incor‑
porated into all future studies of TS genotype, particularly in
relation to its predictive value. Stratification of CRC patients
into subgroups defined by TS LOH status is therefore essen‑tial in obtaining clear evidence for a clinical role of the TS
genotype.
Acknowledgements
The authors would like to thank Dr. Barry Iacopetta(School of Surgery, University of Western Australia) for thecritical reading of the manuscript and for providing valuablesuggestions.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current study areavailable from the corresponding author on reasonable request.

7

Authors' contributions
MKo and KK conceived and designed the current study.
MKo, HB, MKa and KK collected clinical samples. MKo,
HB, MKa, HT and KK performed the experiments andanalyzed the data. MKo, TM and KK drafted the manu‑
script. MKo and TM confirmed the authenticity of all theraw data. All authors read and approved the final version ofmanuscript.
Ethics approval and consent to participate
The current study was performed in accordance with the
Declaration of Helsinki. Since tissues used in this study wereobtained from the patients diagnosed between 1999 and2010, written informed consent was available for most but
not all patients. However in accordance with Japanese ethicalguidelines and law, the study protocol was reviewed andapproved by the Kanazawa University Human Genome/GeneAnalysis Research Ethics Committee (approval nos. 181
and 264). Following instruction by the Ethics Committee atapproval, all patients were publicly provided with an oppor‑
tunity to opt‑out from registration to the current study. Nonedeclined. All samples were anonymized before analysis was
performed to guarantee the protection of privacy.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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