JP5130465B2 - Hepatocellular carcinoma marker and method for testing hepatocellular carcinoma - Google Patents

Hepatocellular carcinoma marker and method for testing hepatocellular carcinoma Download PDF

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JP5130465B2
JP5130465B2 JP2007332596A JP2007332596A JP5130465B2 JP 5130465 B2 JP5130465 B2 JP 5130465B2 JP 2007332596 A JP2007332596 A JP 2007332596A JP 2007332596 A JP2007332596 A JP 2007332596A JP 5130465 B2 JP5130465 B2 JP 5130465B2
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hepatocellular carcinoma
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JP2009156627A (en
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格 近藤
達也 折茂
英知 尾島
説雄 廣橋
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney

Description

本発明は、肝細胞癌の予後を判断するための新規肝細胞癌マーカー及び当該マーカーを用いた肝細胞癌の検査方法に関する。より詳しくは、肝細胞癌を患う患者の予後を予測するための検査方法に関し、さらには検査のための検査用試薬キットに関する。   The present invention relates to a novel hepatocellular carcinoma marker for determining the prognosis of hepatocellular carcinoma and a method for examining hepatocellular carcinoma using the marker. More specifically, the present invention relates to a test method for predicting the prognosis of a patient suffering from hepatocellular carcinoma, and further relates to a test reagent kit for testing.

肝細胞癌（ＨＣＣ）は世界的に一般的かつ悪性度の高い悪性腫瘍のうちの１つで、癌による死因で３番目に挙げられる。肝細胞癌はアジアとアフリカでは大きな健康問題となっている。また、最近の研究によると肝細胞癌の発生率は、最近の数十年間の間で米国と英国で増加しているようである。   Hepatocellular carcinoma (HCC) is one of the most common and high-grade malignant tumors worldwide and is the third leading cause of cancer death. Hepatocellular carcinoma is a major health problem in Asia and Africa. Also, according to recent studies, the incidence of hepatocellular carcinoma appears to have increased in the United States and the United Kingdom over the last few decades.

癌の診断にはＸ線ＣＴやＭＲＩなどの画像診断のほか、特定の癌に特異的に発現する癌マーカーや血液、組織中に漏出する癌マーカーなどを検出する方法も汎用されている。肝癌のマーカーとしては、ＡＦＰ(α-fetoprotein)やＰＩＶＫＡ-II (protein induced by vitamin K absence or antagonist-II)などが知られている。   In addition to diagnostic imaging such as X-ray CT and MRI, methods for detecting cancer markers that are specifically expressed in specific cancers, cancer markers that leak into blood and tissues, and the like are also widely used for cancer diagnosis. Known markers for liver cancer include AFP (α-fetoprotein) and PIVKA-II (protein induced by vitamin K absence or antagonist-II).

肝細胞癌は、化学療法や放射線治療に抵抗性を示し、手術が唯一の完全寛解療法とされてきた。予後不良症例を事前に予測することができれば、手術以外の治療方法や、手術後の治療についてもより効果的な治療方法を選択し、過度な治療を避けることができる。このような予後予測マーカーとして実用化のレベルに至ったものはない。   Hepatocellular carcinoma is resistant to chemotherapy and radiation therapy, and surgery has been the only complete remission therapy. If a case with a poor prognosis can be predicted in advance, it is possible to select a treatment method other than surgery or a more effective treatment method for treatment after surgery, and avoid excessive treatment. No such prognostic marker has reached a practical level.

なお、癌マーカーにおいては、診断マーカー、予後予測マーカー、治療奏効性予測マーカーなどの種類が挙げられる。例えば、ＥＧＦＲキナーゼ阻害剤に対する感受性を予測する治療奏効性予測マーカーとして、KCIP-1, CD98, DRF1, LASP-1, APC-binding protein EB1など各種マーカーが開示されている報告がある（特許文献１）。しかしながら、特許文献１に記載される治療奏効性予測マーカーは、薬剤に関連するマーカーであって、本明細書に記載される癌患者の予後を予測するマーカーとは検査の目的や用途が全く異なるものである。   Examples of cancer markers include diagnostic markers, prognostic prediction markers, and therapeutic response prediction markers. For example, there are reports that various markers such as KCIP-1, CD98, DRF1, LASP-1, and APC-binding protein EB1 are disclosed as predictive markers for therapeutic efficacy for predicting sensitivity to EGFR kinase inhibitors (Patent Document 1). ). However, the therapeutic response prediction marker described in Patent Document 1 is a marker related to a drug, and the purpose and use of the test are completely different from the marker for predicting the prognosis of a cancer patient described in this specification. Is.

肝細胞癌の予後については、癌細胞の分化の程度、門脈侵襲の有無などの病理学的所見に応じて判断するのが一般的である。特に低分化型肝細胞癌のほうが高分化型細胞癌に比べて、予後が悪い傾向がある。しかしながら、生検により得られた検体について癌細胞の分化程度を判断するのは、必ずしも容易ではなく、また肝細胞癌の分化程度と予後について、明確に解明されているわけではない。肝細胞癌に関し、これまで有効な予後予測マーカーについては全く報告されていなかったが、予後を予測しうるマーカーは、癌診断後の治療方法を選択する上の重要な判断基準となるため、強く望まれている。   The prognosis of hepatocellular carcinoma is generally determined according to pathological findings such as the degree of differentiation of cancer cells and the presence or absence of portal vein invasion. In particular, poorly differentiated hepatocellular carcinoma tends to have a worse prognosis than well differentiated cell carcinoma. However, it is not always easy to determine the degree of differentiation of cancer cells in a specimen obtained by biopsy, and the degree of differentiation and prognosis of hepatocellular carcinoma is not clearly elucidated. There have been no reports of effective prognostic markers for hepatocellular carcinoma, but markers that can predict prognosis are important criteria for selecting treatment methods after cancer diagnosis. It is desired.

なお、ＥＢ１はEnd-binding protein 1又はAPC-binding protein EB1と呼ばれるものであり、腫瘍抑制遺伝子産物APC（adenomatous polyposis coli）に結合するタンパク質として発見された公知のタンパク質である（非特許文献１; LK Su et al 1995）。これまでにＥＢ１は肝細胞癌組織（非特許文献２；Fujii et al 2005）、食道癌組織（非特許文献３；Wang et al 2005）、胃癌組織（非特許文献４；Nishigaki et al 2005）において高発現していることが報告されている。しかしながら、予後の予測に関し、ＥＢ１の単独マーカーとしての利用はどの腫瘍についても報告されていない。特に肝細胞癌におけるＥＢ１の予後予測能に関するものは何ら報告されていない。
US2007/0212738米国出願 Su LK, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R, Trent J, Vogelstein B,Kinzler KW. APC binds to the novel protein EB1 Cancer Res. 1995 Jul 15;55(14):2972-7. Fujii K, Kondo T, Yokoo H, Yamada T, Iwatsuki K, Hirohashi S. Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye. Proteomics. 2005 Apr;5(5):1411-22. Wang Y, Zhou X, Zhu H, Liu S, Zhou C, Zhang G, Xue L, Lu N, Quan L, Bai J, Zhan Q, Xu N. Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin/TCF pathway. Oncogene. 2005 Oct 6;24(44):6637-45. Nishigaki R, Osaki M, Hiratsuka M, Toda T, Murakami K, Jeang KT, Ito H, Inoue T, Oshimura M. Proteomic identification of differentially-expressed genes in human gastric carcinomas. Proteomics. 2005 Aug;5(12):3205-13. EB1 is called End-binding protein 1 or APC-binding protein EB1, and is a known protein discovered as a protein that binds to the tumor suppressor gene product APC (adenomatous polyposis coli) (Non-patent Document 1; LK Su et al 1995). So far, EB1 has been used in hepatocellular carcinoma tissue (Non-patent document 2; Fujii et al 2005), esophageal cancer tissue (non-patent document 3; Wang et al 2005), and stomach cancer tissue (non-patent document 4; Nishigaki et al 2005). High expression has been reported. However, for the prediction of prognosis, the use of EB1 as a single marker has not been reported for any tumor. In particular, nothing related to the prognostic ability of EB1 in hepatocellular carcinoma has been reported.
US2007 / 0212738 US application Su LK, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R, Trent J, Vogelstein B, Kinzler KW. APC binds to the novel protein EB1 Cancer Res. 1995 Jul 15; 55 (14): 2972-7. Fujii K, Kondo T, Yokoo H, Yamada T, Iwatsuki K, Hirohashi S. Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye.Proteomics. 2005 Apr; 5 (5): 1411-22. Wang Y, Zhou X, Zhu H, Liu S, Zhou C, Zhang G, Xue L, Lu N, Quan L, Bai J, Zhan Q, Xu N. Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin / TCF pathway. Oncogene. 2005 Oct 6; 24 (44): 6637-45. Nishigaki R, Osaki M, Hiratsuka M, Toda T, Murakami K, Jeang KT, Ito H, Inoue T, Oshimura M. Proteomic identification of differentially-expressed genes in human gastric carcinomas. Proteomics. 2005 Aug; 5 (12): 3205 -13.

本発明は、肝細胞癌の予後を判断するための新規肝細胞癌マーカー及び当該マーカーを用いた肝細胞癌の検査方法を提供することを課題とする。さらには当該マーカーを用いて検査するための検査用試薬キットを提供することを課題とする。   An object of the present invention is to provide a novel hepatocellular carcinoma marker for judging the prognosis of hepatocellular carcinoma and a method for examining hepatocellular carcinoma using the marker. It is another object of the present invention to provide a test reagent kit for testing using the marker.

本発明者らは上記課題を解決するために、生体検体から取得した肝細胞癌を含む各種肝細胞からレーザーマイクロダイセクションを用いて細胞を回収し、タンパク質を抽出した後、蛍光二次元電気泳動法と質量分析装置にて予後に関係するタンパク質を解析した。解析の結果、ＥＢ１(End-binding protein 1)に着目し、ＥＢ１と肝細胞癌患者の予後との関係を調べた結果、ＥＢ１が陽性の場合には陰性の場合に比べて５年生存率が有意に低いことが確認され、ＥＢ１が肝細胞癌の予後判断のために有意な癌マーカーとなりうることを見出し、本発明を完成した。   In order to solve the above-mentioned problems, the present inventors recovered cells from various hepatocytes including hepatocellular carcinoma obtained from a biological specimen using a laser microdissection, extracted proteins, and then performed fluorescence two-dimensional electrophoresis. Proteins related to prognosis were analyzed by the method and mass spectrometer. As a result of analysis, focusing on EB1 (End-binding protein 1), and investigating the relationship between EB1 and the prognosis of patients with hepatocellular carcinoma, the 5-year survival rate is higher when EB1 is positive than when negative. It was confirmed that it was significantly low, and EB1 was found to be a significant cancer marker for determining the prognosis of hepatocellular carcinoma, and the present invention was completed.

すなわち、本発明は以下よりなる。
１．生体から生体検体を分離し、当該生体検体中のＥＢ１(End-binding protein 1)を癌予後予測マーカーとして検出又は定量することを特徴とする肝細胞癌の検査方法。
２．生体検体が、肝細胞癌を含む生体検体である前項１に記載の検査方法。
３．ＥＢ１の検出又は定量を、免疫学的手法により行う前項１又は２に記載の検査方法。
４．免疫学的手法が、免疫組織染色である前項３に記載の検査方法。
５．生体検体中のＥＢ１を検出又は定量するための抗ＥＢ１抗体を含む前項１〜４のいずれか１に記載の検査方法に用いる検査用試薬。
６．生体検体中のＥＢ１を検出又は定量するための抗ＥＢ１抗体と、ＥＢ１と抗ＥＢ１抗体の反応を検出しうる標識を含む前項１〜４のいずれか１に記載の検査方法に用いる検査用試薬キット。 That is, this invention consists of the following.
1. A method for examining hepatocellular carcinoma, comprising separating a biological specimen from a living body and detecting or quantifying EB1 (End-binding protein 1) in the biological specimen as a cancer prognosis prediction marker.
2. 2. The examination method according to item 1 above, wherein the biological specimen is a biological specimen containing hepatocellular carcinoma.
3. 3. The test method according to item 1 or 2, wherein the detection or quantification of EB1 is performed by an immunological technique.
4). 4. The examination method according to item 3 above, wherein the immunological technique is immunohistological staining.
5. 5. A test reagent used in the test method according to any one of 1 to 4 above, comprising an anti-EB1 antibody for detecting or quantifying EB1 in a biological sample.
6). The reagent kit for a test | inspection used for the test | inspection method of any one of the preceding clauses 1-4 containing the label | marker which can detect the reaction of EB1 and an anti- EB1 antibody, and the anti- EB1 antibody for detecting or quantifying EB1 in a biological sample .

本発明の肝細胞癌の検査方法によると、肝癌細胞がＥＢ１陽性症例での術後５年生存率は２２．６％であったのに対し、ＥＢ１陰性症例では６０．９％であり、陽性症例は陰性症例に比べて有意に予後不良であった。更に、ＥＢ１と肝細胞癌の予後に相関される他の臨床病理学因子について多変量解析により解析した結果、術後生存、術後再発ともにＥＢ１は肝細胞癌の予後判断のための強力なマーカーとなることが確認された。   According to the method for examining hepatocellular carcinoma of the present invention, the 5-year survival rate after surgery for hepatoma cells in EB1 positive cases was 22.6%, whereas in EB1 negative cases, it was 60.9%. Cases had a significantly worse prognosis than negative cases. Furthermore, as a result of multivariate analysis of EB1 and other clinicopathological factors correlated with the prognosis of hepatocellular carcinoma, EB1 is a powerful marker for prognosis of hepatocellular carcinoma in both postoperative survival and postoperative recurrence It was confirmed that

ＥＢ１を単一のマーカーとして用いる本発明の肝細胞癌の検査方法によると、肝細胞癌患者の予後を予測することが可能になる。肝細胞癌と診断された患者について、生検などで肝癌細胞を採取した際や手術で切除された肝細胞癌検体を、本発明の検査方法により検査することで、予後を判断することができ、適切な治療方法を選択することができる。つまり、ＥＢ１を単一のマーカーとして用いることにより、技術的に簡便に迅速に低いコストで実用的に肝細胞癌患者の予後を予測し、治療方針を立てることが可能になる。   According to the test method for hepatocellular carcinoma of the present invention using EB1 as a single marker, it becomes possible to predict the prognosis of patients with hepatocellular carcinoma. For patients diagnosed with hepatocellular carcinoma, the prognosis can be determined by examining hepatocellular carcinoma specimens collected by biopsy or by removing surgically removed hepatocellular carcinoma cells by the examination method of the present invention. Appropriate treatment methods can be selected. That is, by using EB1 as a single marker, it is possible to predict the prognosis of a hepatocellular carcinoma patient practically at a low cost in a technically simple and rapid manner, and to make a treatment policy.

本発明の肝細胞癌の検査方法は、癌マーカーとしてＥＢ１を検出又は定量することによる。本発明におけるＥＢ１とは、上記背景技術の欄で示したように公知のタンパク質であり特に限定されないが、例えば非特許文献１〜４に示されるタンパク質をいう。そのアミノ酸配列は、出願時において、Swissprot detabase Accession No. Q15691に掲載されているものを一例としてあげることができるが、いわゆるＥＢ１タンパク質であれば、上記掲載されている配列からなるタンパク質に限定されるものではない。   The test method for hepatocellular carcinoma of the present invention is based on detecting or quantifying EB1 as a cancer marker. EB1 in the present invention is a known protein as shown in the background art section and is not particularly limited, but refers to, for example, the proteins shown in Non-Patent Documents 1 to 4. As for the amino acid sequence, those listed in Swisssprot detabase Accession No. Q15691 at the time of filing can be mentioned as an example, but so-called EB1 protein is limited to the protein consisting of the above-mentioned sequence. It is not a thing.

本発明において、ＥＢ１に着目した経緯は、以下の検討による。国立がんセンター病院から倫理委員会の承諾を得て合計４５の生体検体を得た。これらの検体には、（１）正常肝７症例、（２）肝細胞癌患者の非癌肝（背景肝）１１症例、（３）高分化型肝細胞癌６例、（４）中分化型肝細胞癌１４例、（５）低分化型肝細胞癌７例の肝細胞を含む。上記肝細胞を含む生体検体について、レーザーマイクロダイセクションを用いて肝細胞を回収し、タンパク質を抽出した後、蛍光二次元電気泳動(2D-DIGE)と質量分析装置にて予後に関係するタンパク質を解析した。   In the present invention, the background focused on EB1 is based on the following examination. A total of 45 biological specimens were obtained with the approval of the Ethics Committee from the National Cancer Center Hospital. These specimens included (1) normal liver 7 cases, (2) non-cancerous liver (background liver) 11 cases of hepatocellular carcinoma patients, (3) well differentiated hepatocellular carcinoma 6 cases, (4) moderately differentiated type 14 cases of hepatocellular carcinoma, (5) hepatocytes of 7 cases of poorly differentiated hepatocellular carcinoma. For biological specimens containing the above hepatocytes, hepatocytes are collected using laser microdissection, proteins are extracted, and proteins related to prognosis are analyzed using fluorescence two-dimensional electrophoresis (2D-DIGE) and mass spectrometry. Analyzed.

これにより得られた、約5000個のタンパク質スポットから、各群間の比較において、（１）Wilcoxon検定 p<0.01、（２）平均値の値が３倍以上の差、（３）ｐ値の低い順番に上位２０番目まで、という基準でタンパク質スポット１２０個を選別した。質量分析装置を用いたタンパク質同定の結果、ＥＢ１に由来するタンパク質スポットが、１２０個のスポット中に含まれていた。このＥＢ１に由来するタンパク質スポットの濃度は、正常肝、背景肝、高分化型肝細胞癌では低かったが、中分化型肝細胞癌、低分化型肝細胞癌では高い傾向にあった（図１、２）。そこで、ＥＢ１と肝細胞癌との関係について解析を行った結果、ＥＢ１の発現の有無により肝細胞癌の予後に影響を及ぼすことが確認された。   From the approximately 5000 protein spots obtained in this way, in the comparison between each group, (1) Wilcoxon test p <0.01, (2) difference of the average value more than 3 times, (3) p-value 120 protein spots were selected on the basis of the top 20 in descending order. As a result of protein identification using a mass spectrometer, protein spots derived from EB1 were included in 120 spots. The concentration of the protein spot derived from EB1 was low in normal liver, background liver, and well-differentiated hepatocellular carcinoma, but tended to be high in moderately-differentiated hepatocellular carcinoma and poorly differentiated hepatocellular carcinoma (FIG. 1). 2). Thus, as a result of analyzing the relationship between EB1 and hepatocellular carcinoma, it was confirmed that the presence or absence of expression of EB1 affects the prognosis of hepatocellular carcinoma.

本明細書において、生体から取得した検体を生体検体といい、各種検査や試験に供するために前処理された検体を試料ということとする。本発明の肝細胞癌の検査方法に供するための生体検体は、肝細胞癌患者の組織や血液に由来するものであればよく、好ましくは肝癌細胞を含む検体であれば良い。生体検体は、患者から採取された検体であり、本発明の検査方法のために取得した検体であってもよいが、他の検査に供するために取得した検体や、手術により採取した検体であってもよい。例えば検体を免疫組織染色検査に供する場合、検査に供する試料として、患者から得られた検体から調製したパラフィン切片を用いることができる。また、例えば検体をウェスタンブロット法又はＲＴ−ＰＣＲに供する場合、試験に供する試料として、患者から得られた検体から調製したタンパク質抽出液又はｍＲＮＡ抽出液を用いることができる。   In this specification, a specimen obtained from a living body is referred to as a biological specimen, and a specimen pretreated for use in various tests and tests is referred to as a sample. The biological specimen for use in the method for examining hepatocellular carcinoma of the present invention may be any specimen derived from tissue or blood of a hepatocellular carcinoma patient, preferably a specimen containing hepatoma cells. A biological sample is a sample collected from a patient and may be a sample obtained for the examination method of the present invention, but is a sample obtained for use in another examination or a specimen collected by surgery. May be. For example, when a specimen is subjected to an immunohistochemical staining test, a paraffin section prepared from a specimen obtained from a patient can be used as a sample to be subjected to the examination. For example, when the specimen is subjected to Western blotting or RT-PCR, a protein extract or mRNA extract prepared from a specimen obtained from a patient can be used as a sample to be tested.

本発明の肝細胞癌の検査方法において、ＥＢ１を癌マーカーとして検出又は定量する方法は、生体検体中のＥＢ１を確認可能な方法であれば良く、特に限定されない。各生体検体におけるＥＢ１は、以下に例示する通り任意の方法で検出又は定量することができる。なお、ＥＢ１の検出又は定量は、単にＥＢ１の有無を検出するものであってもよく、またＥＢ１の発現量を相対的又は絶対的に決定するものでもよい。ＥＢ１発現は、タンパク質レベルで検出又は定量してもよく、またｍＲＮＡレベルで検出又は定量してもよい。   In the method for examining hepatocellular carcinoma of the present invention, the method for detecting or quantifying EB1 as a cancer marker is not particularly limited as long as it can confirm EB1 in a biological sample. EB1 in each biological specimen can be detected or quantified by an arbitrary method as exemplified below. The detection or quantification of EB1 may be simply detection of the presence or absence of EB1, or may determine the expression level of EB1 relative or absolute. EB1 expression may be detected or quantified at the protein level, or may be detected or quantified at the mRNA level.

ＥＢ１発現のタンパク質レベルでの検出又は定量は、免疫学的手法によるのが簡便であり、好適である。例えば、免疫染色法（蛍光抗体法、酵素抗体法、重金属標識抗体法、放射性同位元素標識抗体法を含む）、電気泳動法による分離と蛍光、酵素、放射性同位元素などによる検出又は定量との組み合わせ（ウェスタンブロット法、蛍光二次元電気泳動法を含む）、酵素免疫測定吸着法（ＥＬＩＳＡ）、ドット・ブロッティング法等により行うことができる。また、ｍＲＮＡレベルでの検出又は定量は、例えば、ＲＴ−ＰＣＲ（好ましくはリアルタイムＲＴ−ＰＣＲ）、ノーザン・ブロッティング法、Branched DNAアッセイ等により行うことができる。   The detection or quantification of EB1 expression at the protein level is preferably performed conveniently by an immunological technique. For example, immunostaining method (including fluorescent antibody method, enzyme antibody method, heavy metal labeled antibody method, radioisotope labeled antibody method), separation by electrophoresis and detection or quantification by fluorescence, enzyme, radioisotope, etc. (Including Western blotting and fluorescent two-dimensional electrophoresis), enzyme immunoassay adsorption (ELISA), dot blotting and the like. The detection or quantification at the mRNA level can be performed by, for example, RT-PCR (preferably real-time RT-PCR), Northern blotting, Branched DNA assay, and the like.

免疫組織染色法は自体公知の方法を採用することができ、特に限定されないが、その具体例を以下に示す。肝細胞癌患者から分離した生体検体を常法によりホルマリン固定をした後、パラフィンに包埋をしてミクロトームにて厚さ４μｍ程度の組織片に薄切し、スライドガラスに貼り付けたものを切片試料として使用する。切片試料はキシレン処理で完全にパラフィンを除き、１００％から徐々に濃度を下げたアルコール溶液にくぐらせ親水化し水洗する。その後、抗体の浸透性を高めるために耐熱ガラス容器に入れたｐＨ６．０のクエン酸緩衝液中に切片試料を漬け、オートクレーブにて１２１℃で１０分間熱処理し抗原を賦活化する。室温まで放置し冷却し流水で緩衝液を水洗後、ＣＳＡ２キット (catalyzed signal amplification (CSA) system(DAKO社)を用い、免疫組織染色を行う。内因性ペルオキシダーゼ活性、非特異的反応をブロッキングした後、切片試料を抗ＥＢ１抗体を滴下し常温で１５分間反応させる。洗浄後、ＨＲＰ標識抗ウサギ抗体(DAKO社)、増幅試薬及び抗蛍光ＨＲＰ(DAKO社)を用いてそれぞれ１５分間反応させる。洗浄後、ＤＡＢ溶液 (3,3'-diaminobenzidine tetrahydrochloride)(DAKO社)を用いて発色を行う。なお洗浄にはＴＢＳＴ(DAKO社)を用いる。流水にて洗浄後、ヘマトキシリン液にて検体の細胞核を染色する。流水にて水洗後、アルコール溶液、次いでキシレン溶液をくぐらせ脱水し、検体上に封入剤を滴下しカバーグラスを被せて顕微鏡にて観察する。顕微鏡下では肝細胞癌の細胞のＥＢ１タンパク質は茶褐色の発色として観察される。以下に説明するように、その発色により陽性陰性の判定を行うことができる。   The immunohistological staining method can employ a method known per se and is not particularly limited, but specific examples thereof are shown below. A biological specimen isolated from a patient with hepatocellular carcinoma was fixed in formalin by a conventional method, then embedded in paraffin, sliced into tissue pieces of about 4 μm in thickness with a microtome, and sliced on a glass slide Use as a sample. The section sample is completely treated with xylene to remove paraffin, passed through an alcohol solution gradually reduced in concentration from 100%, hydrophilized and washed with water. Thereafter, in order to increase the permeability of the antibody, the section sample is dipped in a pH 6.0 citrate buffer solution placed in a heat-resistant glass container, and the antigen is activated by heat treatment at 121 ° C. for 10 minutes in an autoclave. Allow to cool to room temperature, wash the buffer with running water, and perform immunohistochemical staining using the CSA2 kit (catalyzed signal amplification (CSA) system (DAKO)) After blocking endogenous peroxidase activity and non-specific reactions Then, the anti-EB1 antibody is dropped into the section sample and allowed to react at room temperature for 15 minutes, and then washed for 15 minutes using an HRP-labeled anti-rabbit antibody (DAKO), an amplification reagent and anti-fluorescent HRP (DAKO). Then, color is developed using DAB solution (3,3'-diaminobenzidine tetrahydrochloride) (DAKO), and TBST (DAKO) is used for washing.After washing with running water, the cell nucleus of the specimen is washed with hematoxylin solution. After washing with running water, pass through the alcohol solution and then the xylene solution to dehydrate, drop the mounting medium onto the specimen, cover with a cover glass, and observe under a microscope. The EB1 protein is observed as brownish brown color, and as described below, positive / negative can be determined by the color development.

ＥＢ１発現の検出又は定量の結果は、２種類の段階（陽性及び陰性）に分類することができる。ＥＢ１発現の分類は、検出又は定量方法に応じて、十分な経験を有する病理医、臨床医、検査技師又は検査施設が行うことが好ましい。例えば、ＥＢ１発現の分類は、免疫組織染色法を用いる場合は病理医が行うことができ、ＲＴ−ＰＣＲを用いる場合は検査技師が行うことができる。   The results of detection or quantification of EB1 expression can be classified into two stages (positive and negative). The classification of EB1 expression is preferably performed by a pathologist, clinician, laboratory technician or laboratory with sufficient experience, depending on the detection or quantification method. For example, the classification of EB1 expression can be performed by a pathologist when using an immunohistochemical staining method, and can be performed by a laboratory technician when using RT-PCR.

なお、ＥＢ１発現の分類は、患者からの生体検体におけるＥＢ１の発現量を、コントロールにおけるＥＢ１の発現量と比較することにより行うことが好ましい。ＥＢ１発現の結果を分類する段階の数に応じて、複数のコントロールを用いることが好ましい。例えば、ＥＢ１発現の結果を２種類の段階（陽性及び陰性）に分類する場合は、それぞれの段階に対応した２種類のコントロール（ＥＢ１陽性コントロール及びＥＢ１陰性コントロール）を用いることが好ましい。また、コントロールの１つとして、健常者又は予後良好な肝細胞癌患者に由来するコントロールを用いることが好ましい。   The classification of EB1 expression is preferably performed by comparing the expression level of EB1 in a biological sample from a patient with the expression level of EB1 in a control. It is preferred to use multiple controls depending on the number of stages that classify the results of EB1 expression. For example, when the results of EB1 expression are classified into two types (positive and negative), it is preferable to use two types of controls (EB1 positive control and EB1 negative control) corresponding to each step. As one of the controls, it is preferable to use a control derived from a healthy person or a hepatocellular carcinoma patient with a good prognosis.

免疫組織染色法により、ＥＢ１発現の結果を陽性及び陰性の２種類の段階に分類する場合、免疫組織染色の結果を例えば以下のように判定することができる。肝細胞癌の細胞の細胞質の染色強度を、「陽性染色あり」及び「染色なし」の２段階に分類し、ＥＢ１陽性と陰性の判定をする。癌細胞の半分以上が染色性を示す場合、陽性と判定することができる。   When the result of EB1 expression is classified into two types of positive and negative by the immunohistochemical staining method, the result of immunohistochemical staining can be determined as follows, for example. The cytoplasmic staining intensity of hepatocellular carcinoma cells is classified into two stages, “with positive staining” and “without staining”, and EB1 positive and negative are determined. If more than half of the cancer cells show staining properties, it can be determined as positive.

本発明は、肝細胞癌を患う患者の予後を予測するための検査用試薬並びに検査用試薬キットも含まれる。当該キットにより、患者から得られた生体検体におけるＥＢ１の発現を検出又は定量することができる。すなわち、タンパク質レベルでＥＢ１の発現を検出又は定量するための検査用試薬キットとして、免疫学的手法、例えば免疫組織染色やウェスタンブロット法などに使用される検査用キットが挙げられる。免疫学的手法により検査を行う場合には、少なくとも抗ＥＢ１抗体が検査用試薬に含まれる。抗ＥＢ１抗体は、ＥＢ１の発現を検出しうる抗体であればよく、特に限定されないが、例えばモノクローナル及びポリクローナル抗体、標識化抗体、キメラ抗体、ヒト化抗体ならびにこれらの結合活性断片などが挙げられる。また検査用試薬キットには、上記抗体のほか検出用に用いる標識を含んでいてもよい。キットには、緩衝液、発色基質、二次抗体、ブロッキング剤等の試薬、試験に必要な器具やコントロール等を含むことができる。   The present invention also includes a test reagent and a test reagent kit for predicting the prognosis of a patient suffering from hepatocellular carcinoma. With this kit, the expression of EB1 in a biological specimen obtained from a patient can be detected or quantified. That is, as a test reagent kit for detecting or quantifying the expression of EB1 at the protein level, a test kit used for immunological techniques such as immunohistological staining and Western blotting can be mentioned. When the test is performed by an immunological technique, at least an anti-EB1 antibody is included in the test reagent. The anti-EB1 antibody is not particularly limited as long as it can detect the expression of EB1, and examples thereof include monoclonal and polyclonal antibodies, labeled antibodies, chimeric antibodies, humanized antibodies, and binding activity fragments thereof. Moreover, the test reagent kit may contain a label used for detection in addition to the antibody. The kit can contain a buffer, a chromogenic substrate, a secondary antibody, a reagent such as a blocking agent, instruments and controls necessary for the test, and the like.

以下に本発明の実施例を示し、本発明をさらに具体的に説明するが、本発明はこれらに限定されるものではなく、本発明の技術的思想を逸脱しない範囲内で種々の応用が可能である。   Examples of the present invention will be described below in more detail, but the present invention is not limited to these, and various applications are possible without departing from the technical idea of the present invention. It is.

（実施例１）ウェスタンブロット法によるＥＢ１の確認
正常肝、背景肝、高分化型肝細胞癌、中分化型肝細胞癌、低分化型肝細胞癌を３例ずつ、計１５症例の生体検体について、ＥＢ１に対する特異抗体（抗ＥＢ１抗体）を用いてウェスタンブロット法によりＥＢ１の発現を調べた。各生体検体は手術検体から取得したものであり、国立がんセンターの倫理委員会の審査及び承認を得たものである。
ウェスタンブロット法を行う前に、各生体検体について以下の前処理を行った。 (Example 1) Confirmation of EB1 by Western blotting About 15 cases of biological specimens, 3 cases each of normal liver, background liver, well differentiated hepatocellular carcinoma, moderately differentiated hepatocellular carcinoma, and poorly differentiated hepatocellular carcinoma The expression of EB1 was examined by Western blotting using a specific antibody against EB1 (anti-EB1 antibody). Each biological specimen was obtained from a surgical specimen, and was reviewed and approved by the National Cancer Center Ethics Committee.
Before performing the Western blotting, the following pretreatment was performed on each biological specimen.

１）タンパク質の回収
各症例の手術検体から肝細胞を含む組織を回収し、液体窒素で凍らせた。凍らせた組織をマルチビーズショッカーTM（安井機器、大阪）にて破砕して粉末状にした。粉末状にした組織にタンパク質抽出用緩衝液（６Ｍ ウレア、２Ｍ チオウレア、３％ ＣＨＡＰＳ、１％ TritonX-100）を加えてタンパク質を抽出した。 1) Protein recovery Tissue containing hepatocytes was recovered from surgical specimens of each case and frozen in liquid nitrogen. The frozen tissue was crushed with Multi Bead Shocker ™ (Yasui Kikai, Osaka) to form a powder. Protein extraction buffer (6M urea, 2M thiourea, 3% CHAPS, 1% TritonX-100) was added to the powdered tissue to extract the protein.

２）ウェスタンブロット法
上記抽出したタンパク質２０μｇをＳＤＳ−ＰＡＧＥにて分離し、ニトロセルロース膜に転写した。１次抗体は、ウサギ抗ＥＢ１抗体(Santa Cruz Biotechnology社)を用いた。２次抗体はペルオキシダーゼで標識した抗ウサギ抗体 (GE Healthcare Biosciences社)を用いた。１次抗体は２００倍希釈し、２次抗体は１０００倍希釈した。検出にはＥＣＬキット（GE Healthcare Biosciences社）を使用した。検出したバンドの強度は、ルミノ・イメージアナライザーLAS-3000TM （富士フィルム社）及び ImageQuantTM software (GE Healthcare社)により解析した。 2) Western blotting 20 μg of the extracted protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. Rabbit anti-EB1 antibody (Santa Cruz Biotechnology) was used as the primary antibody. As the secondary antibody, an anti-rabbit antibody (GE Healthcare Biosciences) labeled with peroxidase was used. The primary antibody was diluted 200 times and the secondary antibody was diluted 1000 times. An ECL kit (GE Healthcare Biosciences) was used for detection. The intensity of the detected band was analyzed with Lumino Image Analyzer LAS-3000 ™ (Fuji Film) and ImageQuant ™ software (GE Healthcare).

３）結果
ウェスタンブロット法による解析の結果、蛍光二次元電気泳動法と同様に、正常肝、背景肝、高分化型肝細胞癌では低発現であったが、中分化肝細胞癌、低分化型肝細胞癌では高発現の傾向にあった（図３）。 3) Results As a result of analysis by Western blotting, the expression was low in normal liver, background liver, and well-differentiated hepatocellular carcinoma, as in fluorescence two-dimensional electrophoresis, but moderately differentiated hepatocellular carcinoma and poorly differentiated type. Hepatocellular carcinoma tended to be highly expressed (FIG. 3).

（実施例２）免疫組織染色によるＥＢ１の確認
ＥＢ１の予後予測能を検証する目的で、抗ＥＢ１抗体を用いた免疫組織染色を肝細胞癌１４５症例の検体について行った。 (Example 2) Confirmation of EB1 by immunohistochemical staining For the purpose of verifying the prognostic ability of EB1, immunohistochemical staining using an anti-EB1 antibody was performed on 145 hepatocellular carcinoma specimens.

１）免疫組織染色
パラフィン包埋組織切片試料について、ＣＳＡ染色 (catalyzed signal amplification (CSA) system(DAKO社)の使用説明書の指示に従い、免疫組織染色を行った。通常の方法に従い脱パラフィン処理した切片試料を、１０ｍＭクエン酸塩緩衝液(pH 6.0)中で１０分間１２１℃オートクレーブ滅菌した。切片試料を、５００倍希釈のウサギ抗ＥＢ１ポリクローナル抗体(Santa Cruz社)、ＨＲＰ標識抗ウサギ抗体(DAKO社)、増幅試薬及び抗蛍光ＨＲＰ(DAKO社)を用いて標識した。免疫組織染色のために更にＤＡＢ染色 (3,3'-diaminobenzidine tetrahydrochloride)(DAKO社)を行った。 1) Immunohistochemical staining Paraffin-embedded tissue section samples were subjected to immunohistochemical staining according to the instructions in the instruction manual for CSA staining (catalyzed signal amplification (CSA) system (DAKO)). Section samples were autoclaved in 10 mM citrate buffer (pH 6.0) for 10 minutes at 121 ° C. Section samples were diluted 500-fold with rabbit anti-EB1 polyclonal antibody (Santa Cruz), HRP-labeled anti-rabbit antibody (DAKO). ), Amplification reagent and anti-fluorescent HRP (DAKO) and DAB staining (3,3′-diaminobenzidine tetrahydrochloride) (DAKO) for immunohistological staining.

免疫組織染色によるＥＢ１染色の結果を図４に示す。高分化型癌細胞、中分化型癌細胞及び低分化型癌細胞について確認した。低分化型癌細胞において特に染色が認められた（図４）。ＥＢ１の陽性又は陰性の判断は、２名の熟練者によりなされ、癌細胞のうち、５０％以上がＥＢ１染色されたものをＥＢ１陽性とし、５０％未満のものをＥＢ１陰性と判断した。   The results of EB1 staining by immunohistochemical staining are shown in FIG. Highly differentiated cancer cells, moderately differentiated cancer cells and poorly differentiated cancer cells were confirmed. Staining was particularly observed in poorly differentiated cancer cells (FIG. 4). The judgment of EB1 positive or negative was made by two experts, and among cancer cells, those in which 50% or more were stained with EB1 were regarded as EB1 positive, and those less than 50% were judged as EB1 negative.

２）ＥＢ１の発現と予後の確認
免疫組織染色を行った１４５症例のうち、ＥＢ１陽性症例は４０例、陰性症例は１０５例であった。ＥＢ１陽性症例は中分化肝細胞癌、低分化型肝細胞癌に多い傾向にあった（表１）。予後との関連を検討した結果、術後５年生存率はＥＢ１陽性症例では２２．６％であったのに対し、ＥＢ１陰性症例では６０．９％であり、陽性症例は陰性症例よりも有意に予後不良であった(p<0.0001)（図５Ａ）。また術後５年無再発生存率に関してもＥＢ１陽性症例は７．８％であったのに対し、ＥＢ１陰性症例は２９．９％であり、陽性症例は陰性症例よりも有意に再発が多く認められた(p<0.0001)（図５Ｂ）。 2) Expression of EB1 and confirmation of prognosis Among 145 cases subjected to immunohistochemical staining, 40 cases were positive for EB1 and 105 cases were negative. EB1 positive cases tended to be more common in moderately differentiated hepatocellular carcinoma and poorly differentiated hepatocellular carcinoma (Table 1). As a result of examining the relationship with prognosis, the 5-year survival rate after surgery was 22.6% in EB1-positive cases, whereas it was 60.9% in EB1-negative cases, and positive cases were more significant than negative cases. The prognosis was poor (p <0.0001) (FIG. 5A). In addition, EB1 positive cases were 7.8% in 5 years after surgery, whereas EB1 negative cases were 29.9%, and positive cases showed significantly more recurrence than negative cases. (P <0.0001) (FIG. 5B).

肝細胞癌患者１４５症例のＥＢ１の発現と臨床病理学的背景の関連を表２に示した。ＥＢ１の発現は統計学的に有意な確からしさ（p<0.01）をもってステージ、分化度、門脈侵襲、肝内転移などの肝細胞癌に重要な臨床病理学的背景に相関することが分かった。また、単変量解析と多変量解析を用いて肝細胞癌の予後に相関するとされている他の臨床病理学的因子(門脈浸潤など)もＥＢ１とあわせて予後に対する影響を検討した結果を表３に示した。多変量解析の結果、肝硬変、門脈侵襲、肝内転移、ＥＢ１が独立した予後因子であることが分かった。腫瘍側因子としては、ＥＢ１は術後生存、術後再発に関し最も強力（ハザード比2.289）なマーカーとなりうることが明らかとなった。   Table 2 shows the relationship between EB1 expression and clinicopathological background in 145 hepatocellular carcinoma patients. The expression of EB1 was found to correlate with the clinical pathological background important for hepatocellular carcinoma such as stage, degree of differentiation, portal vein invasion and intrahepatic metastasis with statistically significant probability (p <0.01). . In addition, other clinicopathological factors (such as portal vein invasion) that have been correlated with the prognosis of hepatocellular carcinoma using univariate analysis and multivariate analysis, together with EB1, examined the effect on prognosis. It was shown in 3. As a result of multivariate analysis, it was found that cirrhosis, portal vein invasion, intrahepatic metastasis, and EB1 are independent prognostic factors. As a tumor-side factor, it was revealed that EB1 can be the most powerful marker (hazard ratio 2.289) regarding postoperative survival and postoperative recurrence.

（参考例１）二次元電気泳動及び質量分析による解析
本発明において、ＥＢ１を肝細胞癌予後予測マーカーとして選別するに至った経緯として、二次元電気泳動及び質量分析を行い、肝細胞癌の予後に影響を及ぼすタンパク質を選別した。その内容について、以下に具体的に示す。 (Reference Example 1) Analysis by Two-dimensional Electrophoresis and Mass Spectrometry In the present invention, two-dimensional electrophoresis and mass spectrometry were performed as a background for selecting EB1 as a prognostic marker for hepatocellular carcinoma. Proteins that affect The contents are specifically shown below.

１）試料調製
レーザーマイクロダイセクション（mmi CellCutTM、NIPPN TechnoCluster社）を用いて細胞を回収し、タンパク質抽出用緩衝液（６Ｍ ウレア、２Ｍ チオウレア、３％ ＣＨＡＰＳ、１％ TritonX-100）を加えてタンパク質を抽出した。抽出したタンパク質を蛍光色素（サチュレーションダイＣｙ５TM、GE Healthcare Biosciences社）で標識した。標識は以下のように行った。（１）終濃度３０ｍＭとなるようにｐＨ８．０のトリス緩衝液を加え、次に（２）１ｎｍｏｌのＴＥＣＰ（トリス（２−カルボキシエチル）ホスフィンヒドロクロライド，Sigma社）を加え、（３）３７℃で６０分間処理した。次に、（４）Ｃｙ５蛍光色素を４ｎｍｏｌ加えて、３７℃で３０分間処理した。今回の実験に用いたタンパク質試料から等量ずつタンパク質試料を集めて混合し、内部コントロール試料とした。内部コントロール試料を蛍光色素（サチュレーションダイＣｙ３TM、GE Healthcare Biosciences社）で上記と同様に標識した。Ｃｙ５で標識した個別のサンプルとＣｙ３で標識した内部コントロール試料を混合し、ウレア可溶化液で最終容量４２０μｌとした。その際、終濃度が６５ｍＭとなるようにジチオスレイトール（DDT）を、２％となるようにとアンフォラインTM（GE Healthcare Biosciences社）を加えた。Ｃｙ５で標識した個別試料とＣｙ３で標識した内部コントロール試料を混合したサンプルを一枚の二次元電気泳動ゲルで泳動した。 1) Sample preparation Cells were collected using laser microdissection (mmi CellCut ™ , NIPPN TechnoCluster), and buffer for protein extraction (6M urea, 2M thiourea, 3% CHAPS, 1% TritonX-100) was added. Protein was extracted. The extracted protein was labeled with a fluorescent dye (saturation dye Cy5 ™ , GE Healthcare Biosciences). The labeling was performed as follows. (1) A Tris buffer solution having a pH of 8.0 is added to a final concentration of 30 mM, and then (2) 1 nmol of TECP (Tris (2-carboxyethyl) phosphine hydrochloride, Sigma) is added. (3) 37 Treated for 60 minutes at ° C. Next, (4) 4 nmol of Cy5 fluorescent dye was added and treated at 37 ° C. for 30 minutes. An equal amount of protein sample was collected from the protein sample used in this experiment and mixed to obtain an internal control sample. The internal control sample was labeled with a fluorescent dye (Saturation Dy Cy3 ™ , GE Healthcare Biosciences) as described above. Individual samples labeled with Cy5 and internal control samples labeled with Cy3 were mixed and brought to a final volume of 420 μl with urea solubilizer. At that time, dithiothreitol (DDT) was added to a final concentration of 65 mM, and Ampholine ™ (GE Healthcare Biosciences) was added to a concentration of 2%. A sample in which an individual sample labeled with Cy5 and an internal control sample labeled with Cy3 were mixed was run on a single two-dimensional electrophoresis gel.

２）二次元電気泳動
まず、一次元目の泳動はイモビラインゲル（２４ｃｍ、ｐＩ４−７、GE Healthcare Biosciences社）と、Multiphor IITM（GE Healthcare Biosciences社）を使用した。泳動するタンパク質試料でイモビラインゲルを室温にて一晩膨潤させた。泳動は40000Vhで行った。二次元目の泳動は９−１５％のポリアクリルアミドのグラジエントゲルと、二次元泳動装置を使用した。泳動は泳動装置一台につき１８Ｗで１０時間、１５℃で行った。 2) Two-dimensional electrophoresis First, immobiline gel (24 cm, pI4-7, GE Healthcare Biosciences) and Multiphor II ™ (GE Healthcare Biosciences) were used for the first-dimensional electrophoresis. Immobiline gels were swollen overnight at room temperature with the protein samples to be run. Electrophoresis was performed at 40000 Vh. The second-dimensional electrophoresis used a 9-15% polyacrylamide gradient gel and a two-dimensional electrophoresis apparatus. Electrophoresis was performed at 15 ° C. for 10 hours at 18 W per electrophoresis apparatus.

３）タンパク質検出
泳動終了後は、タンパク質を検出する目的で、ガラス板に挟んだままの状態のゲルをレーザースキャナー（Typhoon TrioTM、GE Healthcare Biosciences社）に載せてスキャンした。 3) Protein detection After the completion of the electrophoresis, for the purpose of detecting the protein, the gel in a state of being sandwiched between glass plates was placed on a laser scanner (Typhoon Trio ™ , GE Healthcare Biosciences) and scanned.

４）発現解析
読み込んだ画像を画像解析ソフトDeCyderTM（GE Healthcare Bio-sciences社）で解析した。 4) Expression analysis The read image was analyzed with image analysis software DeCyder ™ (GE Healthcare Bio-sciences).

５）タンパク質同定
ａ．ゲル内消化法
全自動スポット回収装置ProHunterTM（AsOne社）を用いて、ゲルから９６穴プレートにスポットを回収した。ゲルをメタノールで十分洗浄し、タンパク質分解酵素（トリプシン）で３７℃にて一晩処理した。この処理によってタンパク質はペプチド化される。得られたペプチドは、６０％アセトニトリルにてゲルを洗浄することで回収した。
ｂ．質量分析
ペプチドの質量を測定するためにＬＴＱTM（サーモエレクトロン社）を使用した。タンパク質同定のためのデータベース検索にはMasCotTMを使用した。 5) Protein identification a. In-gel digestion Spots were collected from the gel in 96-well plates using a fully automated spot collection device ProHunter ™ (AsOne). The gel was thoroughly washed with methanol and treated with proteolytic enzyme (trypsin) at 37 ° C. overnight. This treatment results in peptide formation of the protein. The obtained peptide was recovered by washing the gel with 60% acetonitrile.
b. Mass Spectrometry LTQ ™ (Thermo Electron) was used to measure the mass of the peptide. MasCot ™ was used to search the database for protein identification.

６）結果
上記方法により二次元電気泳動を行った結果、約5000個のタンパク質スポットが得られた（図６）。これにより得られた、約5000個のタンパク質スポットから、各群間の比較において、（１）Wilcoxon検定 p<0.01、（２）平均値の値が３倍以上の差、（３）ｐ値の低い順番に上位２０番目まで、という基準でタンパク質スポット１２０個を選別した。質量分析装置を用いたタンパク質同定の結果、ＥＢ１に由来するタンパク質スポットが、１２０個のスポット中に含まれていた。このＥＢ１に由来するタンパク質スポットの濃度は、正常肝、背景肝、高分化型肝細胞癌では低かったが、中分化型肝細胞癌、低分化型肝細胞癌では高い傾向にあった（図１、２）。そこで、ＥＢ１に着目し、ＥＢ１が肝細胞癌の予後予測マーカーとなりうるかについて検討した。これにより、上記実施例２の結果が得られ、予後予測マーカーとして希望しうることが確認できた。 6) Results As a result of performing the two-dimensional electrophoresis by the above method, about 5000 protein spots were obtained (FIG. 6). From the approximately 5000 protein spots obtained in this way, in the comparison between each group, (1) Wilcoxon test p <0.01, (2) difference of the average value more than 3 times, (3) p-value 120 protein spots were selected on the basis of the top 20 in descending order. As a result of protein identification using a mass spectrometer, protein spots derived from EB1 were included in 120 spots. The concentration of the protein spot derived from EB1 was low in normal liver, background liver, and well-differentiated hepatocellular carcinoma, but tended to be high in moderately-differentiated hepatocellular carcinoma and poorly differentiated hepatocellular carcinoma (FIG. 1). 2). Thus, focusing on EB1, whether EB1 could be a prognostic marker for hepatocellular carcinoma was examined. Thereby, the result of the said Example 2 was obtained and it has confirmed that it could hope as a prognosis prediction marker.

以上詳述したように、本発明の肝細胞癌の検査方法によると、肝癌細胞がＥＢ１陽性症例での術後５年生存率は２２．６％であったのに対し、ＥＢ１陰性症例では６０．９％であり、陽性症例は陰性症例に比べて有意に予後不良であった。更に、多変量解析により解析した結果、ＥＢ１と肝細胞癌の予後に相関される他の臨床病理学因子について、術後生存、術後再発ともに相関することが確認され、ＥＢ１は肝細胞癌の予後判断のための強力なマーカーとなることが確認された。   As described in detail above, according to the method for examining hepatocellular carcinoma of the present invention, the 5-year survival rate after surgery for hepatoma cells in EB1 positive cases was 22.6%, whereas in EB1 negative cases, 60%. .9%, and the positive cases had a significantly worse prognosis than the negative cases. Furthermore, as a result of analysis by multivariate analysis, it was confirmed that other clinical pathological factors correlated with the prognosis of EB1 and hepatocellular carcinoma were correlated with both postoperative survival and postoperative recurrence. It was confirmed to be a powerful marker for prognosis judgment.

ＥＢ１を単一のマーカーとして用いる本発明の肝細胞癌の検査方法によると、肝細胞癌患者の予後を予測することが可能になる。肝細胞癌と診断された患者について、生検などで肝癌細胞を採取した際や手術で切除された肝細胞癌検体を、本発明の検査方法により検査することで予後を判断することができ、適切な治療方法を選択することができる。つまり、ＥＢ１を単一のマーカーとして用いることにより、技術的に簡便に迅速に低いコストで実用的に肝細胞癌患者の予後を予測し、治療方針を立てることが可能になる。   According to the test method for hepatocellular carcinoma of the present invention using EB1 as a single marker, it becomes possible to predict the prognosis of patients with hepatocellular carcinoma. For patients diagnosed with hepatocellular carcinoma, the prognosis can be determined by examining the hepatocellular carcinoma specimen resected by surgery when hepatoma cells are collected by biopsy or the like, by examining the examination method of the present invention, Appropriate treatment methods can be selected. That is, by using EB1 as a single marker, it is possible to predict the prognosis of a hepatocellular carcinoma patient practically at a low cost in a technically simple and rapid manner, and to make a treatment policy.

なお、免疫組織染色による手法は、肝細胞癌の分化度を判断するのに比べて検体の取扱いが容易であり、大量の検体を取り扱うことができる。また、予後の予測により適切な治療方法を選択することで、患者に対する経済的、肉体的に過度の負担が軽減化され、医療費の節減にも繋がるものと考えられる。   The technique using immunohistochemical staining is easier to handle than the determination of the degree of differentiation of hepatocellular carcinoma, and can handle a large amount of specimens. In addition, by selecting an appropriate treatment method based on the prognosis, it is considered that an excessive burden on the patient is economically and physically reduced, and the medical cost is reduced.

二次元電気泳動により、正常肝細胞、肝細胞癌患者の非癌細胞（背景肝）、高分化型肝細胞癌、中分化型肝細胞癌及び低分化型肝細胞癌におけるＥＢ１の発現を確認した図である。Two-dimensional electrophoresis confirmed the expression of EB1 in normal hepatocytes, non-cancer cells of hepatocellular carcinoma patients (background liver), well differentiated hepatocellular carcinoma, moderately differentiated hepatocellular carcinoma, and poorly differentiated hepatocellular carcinoma. FIG. 二次元電気泳動により、正常肝細胞、肝細胞癌患者の非癌細胞（背景肝）、高分化型肝細胞癌、中分化型肝細胞癌及び低分化型肝細胞癌におけるＥＢ１の発現量を確認した図である。Two-dimensional electrophoresis confirms the expression level of EB1 in normal hepatocytes, non-cancer cells (background liver) of patients with hepatocellular carcinoma, well differentiated hepatocellular carcinoma, moderately differentiated hepatocellular carcinoma, and poorly differentiated hepatocellular carcinoma FIG. ウェスタンブロット法により、正常肝細胞、肝細胞癌患者の非癌細胞（背景肝）、高分化型肝細胞癌、中分化型肝細胞癌及び低分化型肝細胞癌におけるＥＢ１の発現を確認した図である。（実施例１）Western blotting confirmed the expression of EB1 in normal hepatocytes, non-cancerous cells (background liver) of patients with hepatocellular carcinoma, well differentiated hepatocellular carcinoma, moderately differentiated hepatocellular carcinoma, and poorly differentiated hepatocellular carcinoma It is. Example 1 免疫組織染色により、高分化型肝細胞癌、中分化型肝細胞癌及び低分化型肝細胞癌におけるＥＢ１の発現を確認した図である。（実施例２）It is the figure which confirmed the expression of EB1 in well differentiated hepatocellular carcinoma, moderately differentiated hepatocellular carcinoma, and poorly differentiated hepatocellular carcinoma by immunohistochemical staining. (Example 2) ＥＢ１発現の陽性及び陰性の場合の生存率の違いを示す図である。（実施例２）It is a figure which shows the difference in the survival rate in the case of positive and negative of EB1 expression. (Example 2) 二次元電気泳動による約5000のタンパク質スポットを示す図である。（参考例）It is a figure which shows about 5000 protein spots by two-dimensional electrophoresis. (Reference example)

Claims (6)

分離された生体検体中のＥＢ１(End-binding protein 1)を癌予後予測マーカーとして検出又は定量することを特徴とする肝細胞癌の検査方法。 A test method for hepatocellular carcinoma, which comprises detecting or quantifying EB1 (End-binding protein 1) in a separated biological specimen as a cancer prognosis prediction marker. 生体検体が、肝細胞癌を含む生体検体である請求項１に記載の検査方法。 The test method according to claim 1, wherein the biological specimen is a biological specimen containing hepatocellular carcinoma. ＥＢ１の検出又は定量を、免疫学的手法により行う請求項１又は２に記載の検査方法。 The test method according to claim 1 or 2, wherein the detection or quantification of EB1 is performed by an immunological technique. 免疫学的手法が、免疫組織染色である請求項３に記載の検査方法。 The examination method according to claim 3, wherein the immunological technique is immunohistological staining. 生体検体中のＥＢ１を検出又は定量するための抗ＥＢ１抗体を含む請求項１〜４のいずれか１に記載の肝細胞癌の検査方法に用いる癌予後予測検査用試薬。 The reagent for cancer prognosis prediction test | inspection used for the test | inspection method of hepatocellular carcinoma of any one of Claims 1-4 containing the anti- EB1 antibody for detecting or quantifying EB1 in a biological sample. 生体検体中のＥＢ１を検出又は定量するための抗ＥＢ１抗体と、ＥＢ１と抗ＥＢ１抗体の反応を検出しうる標識を含む請求項１〜４のいずれか１に記載の肝細胞癌の検査方法に用いる癌予後予測検査用試薬キット。 The method for examining hepatocellular carcinoma according to any one of claims 1 to 4, comprising an anti-EB1 antibody for detecting or quantifying EB1 in a biological sample, and a label capable of detecting a reaction between EB1 and the anti-EB1 antibody. Reagent kit for cancer prognosis test used.
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