JP6339814B2 - Cancer metastasis marker and analysis method thereof - Google Patents
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Description
本発明は、基礎医学分野及び臨床医学分野に属し、がん転移関連タンパク質マーカー及びその分析方法に関する。より詳しくは、本発明は、原発がんが存在している状態、或いは存在していたが手術等によって除かれた後に、体内のどこかに転移が起こっていないかどうかの判定に用いるためのがん転移関連タンパク質マーカー及びその分析方法に関する。 The present invention belongs to the fields of basic medicine and clinical medicine, and relates to a cancer metastasis-related protein marker and an analysis method thereof. More specifically, the present invention is for use in determining whether or not metastasis has occurred somewhere in the body after a primary cancer is present or has been present but removed by surgery or the like. The present invention relates to cancer metastasis-related protein markers and analysis methods thereof.
がんは日本人の死亡原因第１位の疾患であり、日本人の３人に１人はがんが原因となって死に至り、２人に１人は生涯に一度はがんに罹患する。また、世界を見渡しても、先進国では同様の傾向となっている。もちろん、がんの治療成績自体は近年向上しており、がんの種類によっては外科的手術による切除で、５年生存率が８０％を越えるものも認められる。しかしながら、転移した場合の治療成績は極めて悪く、実際、がんで亡くなる人の多くが、転移が原因であるとも言われている。がん転移に関連しているタンパク質に関してはいくつか報告があるが、その多くはまだ臨床での実用化には至っていない。 Cancer is the leading cause of death in Japanese people, 1 in 3 Japanese people will die from cancer and 1 in 2 people will have cancer once in their lifetime . Looking at the world, the same tendency is seen in developed countries. Of course, the results of cancer treatment have improved in recent years, and depending on the type of cancer, surgical excision may result in a 5-year survival rate exceeding 80%. However, the treatment results when metastasized are extremely poor, and in fact, many people who die from cancer are said to be caused by metastasis. There are several reports on proteins related to cancer metastasis, but many of them have not yet been put to clinical use.
このような現状において、原発がんが存在している状態、或いは存在していたが手術等によって除かれた後に、体内のどこかに転移が起こっていないかどうかの判定に用いることのできるがん転移関連タンパク質マーカーの開発が望まれる。 Under such circumstances, cancer that can be used to determine whether metastasis has occurred anywhere in the body after the presence of primary cancer or after it has been removed by surgery, etc. Development of metastasis-related protein markers is desired.
特開２０１２−４７５６５号公報（特許文献１）には、大腸がん肝転移マーカー、及びその分析方法が開示され、これらが２−ニトロベンゼンスルフェニルクロリド（ＮＢＳＣｌ）を用いた同位体標識法（ＮＢＳ法）を利用して発明されたことも開示されている（段落［００２７］）。 Japanese Patent Application Laid-Open No. 2012-47565 (Patent Document 1) discloses a liver metastasis marker for colorectal cancer and an analysis method thereof, which uses an isotope labeling method (NBS) using 2-nitrobenzenesulfenyl chloride (NBSCl). (Paragraph [0027]).
Journal of Chromatography B, vol. 877, pp2607-2614 (2009)（非特許文献１）には、2-nitrobenzenesulfenyl（ＮＢＳ）試薬を用いた同位体標識法（ＮＢＳ法）を利用するタンパク質バイオマーカーの検索法が開示されている。 Journal of Chromatography B, vol. 877, pp2607-2614 (2009) (Non-patent Document 1) Search for protein biomarkers using isotope labeling method (NBS method) using 2-nitrobenzenesulfenyl (NBS) reagent The law is disclosed.
International Journal of Oncology, vol. 33, pp741-751 (2007)（非特許文献２）には、「NOD/SCID/γcnull（NOG）マウスにおける転移の新規定量モデルを用いた、ヒト膵臓癌における肝転移の重要な分子調節因子の同定」について開示され、NOGマウスを用いて「がん転移モデル系」を作製することが開示されている。 International Journal of Oncology, vol. 33, pp741-751 (2007) (Non-Patent Document 2) states that "Liver metastasis in human pancreatic cancer using a novel quantitative model of metastasis in NOD / SCID / γcnull (NOG) mice" The identification of an important molecular regulatory factor in the present invention is disclosed, and the creation of a “cancer metastasis model system” using NOG mice is disclosed.
Biochemical and Biophysical Research Communications, vol. 377, pp248-252 (2008)（非特許文献３）には、NOGマウスを用いた肝臓のヒト化モデルの確立について開示されている。 Biochemical and Biophysical Research Communications, vol. 377, pp248-252 (2008) (Non-patent Document 3) discloses the establishment of a humanization model of the liver using NOG mice.
生化学79(7), 643-654, 2007（非特許文献４）には、アクチニン4（actinin-4）の発現と、がん転移・浸潤との関連が報告されている。 Biochemistry 79 (7), 643-654, 2007 (Non-patent Document 4) reports the relationship between the expression of actinin-4 and cancer metastasis / invasion.
Cancer Research,vol. 53(20), pp 4896-9 (1993)（非特許文献５）には、Glucosidase II (β-Glucosidase)の活性と転移との関連が報告されている。 Cancer Research, vol. 53 (20), pp 4896-9 (1993) (Non-patent Document 5) reports the relationship between the activity of Glucosidase II (β-Glucosidase) and metastasis.
Cancer,vol. 58(7),pp1484-7(1986)（非特許文献６）、及びProceedings ofthe National Academy of Sciencesof the USA, vol. 83(8), pp2483-7(1986)（非特許文献７）には、beta-N-acetylhexosaminidase （beta-hexosaminidase/N-acetyl-beta-glucosaminidase）について、血液中等の酵素活性と転移の程度に相関があることが報告されている。 Cancer, vol. 58 (7), pp1484-7 (1986) (Non-patent document 6), and Proceedings of the National Academy of Sciences of the USA, vol. 83 (8), pp2483-7 (1986) (Non-patent document 7) ), It is reported that beta-N-acetylhexosaminidase (beta-hexosaminidase / N-acetyl-beta-glucosaminidase) has a correlation between enzyme activity in blood and the degree of transfer.
原発がんが存在している状態、或いは存在していたが手術等によって除かれた後に、体内のどこかに転移が起こっていないかどうかの判定を行うことができれば、がんの治療成績向上が期待できる。 If the primary cancer is present, or if it was present but removed by surgery, etc., it can be determined whether metastasis has occurred anywhere in the body. I can expect.
そこで、本発明の目的は、原発がんが存在している状態、或いは存在していたが手術等によって除かれた後に、体内のどこかに転移が起こっていないかどうかの判定に用いるためのがん転移関連タンパク質マーカー、及びその分析方法を提供することにある。 Therefore, an object of the present invention is to be used for determining whether or not metastasis has occurred somewhere in the body after the primary cancer is present or has been removed by surgery or the like. It is to provide a metastasis-related protein marker and an analysis method thereof.
本発明者らは、鋭意検討の結果、ＮＯＧマウスを用いて作成した「がん転移モデル系」を利用し、ＮＢＳ試薬含む定量プロテオーム解析を行い、上記目的を達成したがん転移関連タンパク質マーカーを見出し、本発明を完成するに至った。 As a result of intensive studies, the present inventors conducted a quantitative proteomic analysis including an NBS reagent using a “cancer metastasis model system” created using NOG mice, and found a cancer metastasis-related protein marker that achieved the above object. The headline and the present invention were completed.
本発明は、以下の発明を含む。
（１） Zinc finger protein 185、
Cell surface antigen 4F2 heavy chain、
Isocitrate dehydrogenase 1、
Interleukin enhancer-binding factor 3、
Transcription factor IIF, alpha subunit、
Torsin-1A-interacting protein 1、
Activated RNA polymerase II transcriptional coactivator p15、及び
Clathrin heavy chain 1
からなる群から選ばれる少なくとも１種のタンパク質を含む、がん転移マーカー。
The present invention includes the following inventions.
(1) Zinc finger protein 185,
Cell surface antigen 4F2 heavy chain,
Isocitrate dehydrogenase 1,
Interleukin enhancer-binding factor 3,
Transcription factor IIF, alpha subunit,
Torsin-1A-interacting protein 1,
Activated RNA polymerase II transcriptional coactivator p15, and
Clathrin heavy chain 1
A cancer metastasis marker comprising at least one protein selected from the group consisting of:
なお、”Transcription factor IIF, alpha subunit”は、一つのタンパク質名称である。 “Transcription factor IIF, alpha subunit” is one protein name.
（２） 原発がんが大腸がん又は膵臓がんである、上記（１）に記載のがん転移マーカー。 (2) The cancer metastasis marker according to (1), wherein the primary cancer is colon cancer or pancreatic cancer.
（３） 生体試料中の、
Zinc finger protein 185、
Cell surface antigen 4F2 heavy chain、
Isocitrate dehydrogenase 1、
Interleukin enhancer-binding factor 3、
Transcription factor IIF, alpha subunit、
Torsin-1A-interacting protein 1、
Activated RNA polymerase II transcriptional coactivator p15、及び
Clathrin heavy chain 1
からなる群から選ばれる少なくとも１種のがん転移マーカーの発現レベルを取得する工程と、前記がん転移マーカーの基準レベルに基づき、前記発現レベルの高低によって評価を行う工程とを含む、がん転移マーカーの分析方法。
(3) in a biological sample
Zinc finger protein 185,
Cell surface antigen 4F2 heavy chain,
Isocitrate dehydrogenase 1,
Interleukin enhancer-binding factor 3,
Transcription factor IIF, alpha subunit,
Torsin-1A-interacting protein 1,
Activated RNA polymerase II transcriptional coactivator p15, and
Clathrin heavy chain 1
Cancer, comprising: obtaining an expression level of at least one cancer metastasis marker selected from the group consisting of: and evaluating the expression level based on a reference level of the cancer metastasis marker Method for analyzing metastatic markers.
（４） 前記がん転移マーカーの発現レベルの取得工程は、生体試料の画像分析を含む、上記（３）に記載の方法。 (4) The method according to (3), wherein the step of obtaining the expression level of the cancer metastasis marker includes image analysis of a biological sample.
（５） 前記生体試料が免疫組織化学染色されるべきがん患者の原発巣組織切片であり、前記発現レベルが前記組織におけるがん部位の染色強度である、上記（３）又は（４）に記載の方法。 (5) In the above (3) or (4), the biological sample is a primary tissue section of a cancer patient to be immunohistochemically stained, and the expression level is a staining intensity of a cancer site in the tissue The method described.
（６） 前記生体試料が破砕された組織を含むものであり、前記発現レベルが前記試料中において検出された前記がん転移マーカーの測定濃度であり、前記基準レベルが前記がん転移マーカーの基準濃度である、上記（３）に記載の方法。 (6) The biological sample includes a crushed tissue, the expression level is a measured concentration of the cancer metastasis marker detected in the sample, and the reference level is a reference of the cancer metastasis marker The method according to (3) above, which is a concentration.
前記（４）〜（６）に記載の方法において、前記組織としては、生検組織（組織生検材料）でありうる。生検組織は、検査・手術などで採取された組織である。生検組織としては、切除組織でありうる。切除組織は、手術で切除された組織である。 In the methods according to (4) to (6), the tissue may be a biopsy tissue (tissue biopsy material). A biopsy tissue is a tissue collected by examination or surgery. The biopsy tissue can be a resected tissue. The excised tissue is tissue that has been excised by surgery.
（７） 前記生体試料ががん患者の血液であり、前記発現レベルが血中におけるがん転移マーカーの発現強度である、上記（３）に記載の方法。前記方法において、採取された血液試料は、生体内に戻されることなく破棄される。 (7) The method according to (3), wherein the biological sample is blood of a cancer patient, and the expression level is an expression intensity of a cancer metastasis marker in blood. In the method, the collected blood sample is discarded without being returned to the living body.
本発明により、生体内におけるがん転移関連タンパク質マーカーが提供される。 The present invention provides a cancer metastasis-related protein marker in vivo.
また、本発明のがん転移マーカーの提供により、生体から採取した試料を用いて、原発がんが存在している状態、或いは存在していたが手術等によって除かれた後に、体内のどこかへの転移の有無を判定するための分析方法を提供することができる。この分析方法により、転移がんの早期発見を行うことができ、また、原発がんが転移を起こしやすいがんなのか否かの判定を行うことができる。 In addition, by providing the cancer metastasis marker of the present invention, using a sample collected from a living body, the state where the primary cancer is present, or after having been removed by surgery or the like, to somewhere in the body An analysis method for determining the presence or absence of metastasis can be provided. By this analysis method, early detection of metastatic cancer can be performed, and it can be determined whether or not the primary cancer is a cancer that easily causes metastasis.
転移がんの早期発見により、転移がんの早期治療を行うことができ、がんの治療成績向上につながる。 Early detection of metastatic cancer allows early treatment of metastatic cancer, leading to improved cancer treatment results.
［がん転移タンパク質マーカー］
本発明は、がん転移タンパク質マーカーを提供する。がん転移タンパク質マーカーの探索は、以下のようにして行うことができる。
[Cancer metastasis protein marker]
The present invention provides a cancer metastasis protein marker. The search for a cancer metastasis protein marker can be performed as follows.
前述の非特許文献２に従って、ＮＯＧマウスを用いて作成した「がん転移モデル系」を利用してヒトの「がん高転移細胞株」を樹立する。次に、この「がん高転移細胞株」と元の「親細胞株」とを用いて、前述の非特許文献１等に開示されている２−ニトロベンゼンスルフェニルクロリド（ＮＢＳ）試薬を用いた同位体標識法（ＮＢＳ法）解析システムを利用して、定量プロテオーム解析を行う。この手法により、がん転移に関連する多数のタンパク質（以下、「がん転移関連タンパク質」と記載する）を同定することができる。 In accordance with the above-mentioned Non-Patent Document 2, a human “cancer metastasis cell line” is established using a “cancer metastasis model system” created using NOG mice. Next, using this “cancer highly metastatic cell line” and the original “parent cell line”, the 2-nitrobenzenesulfenyl chloride (NBS) reagent disclosed in the aforementioned Non-Patent Document 1 or the like was used. Quantitative proteome analysis is performed using an isotope labeling (NBS) analysis system. By this technique, a large number of proteins related to cancer metastasis (hereinafter referred to as “cancer metastasis-related proteins”) can be identified.
図１は、ＮＢＳ法解析システムを利用した定量プロテオーム解析の手順を示すフロー図である。図２は、本発明で実施された定量プロテオーム解析におけるさらに詳細なフロー図である。図１及び図２は、膵臓がん細胞株である「ＢｘＰＣ−３」の場合を例として図示されている。 FIG. 1 is a flowchart showing the procedure of quantitative proteome analysis using the NBS method analysis system. FIG. 2 is a more detailed flow diagram in the quantitative proteome analysis performed in the present invention. 1 and 2 illustrate the case of “BxPC-3”, which is a pancreatic cancer cell line, as an example.
図１において、膵臓がん由来細胞株である「ＢｘＰＣ−３」からＮＯＧマウスを用いて膵臓がん高転移細胞株である「ＬＭ−ＢｘＰＣ−３」を作成する。ＢｘＰＣ−３及びＬＭ−ＢｘＰＣ−３をそれぞれ培養し、タンパク質抽出を行う。抽出されたタンパク質試料ＢｘＰＣ−３及びＬＭ−ＢｘＰＣ−３から、２−ニトロベンゼンスルフェニルクロリド（ＮＢＳＣｌ）を用いた同位体標識法（ＮＢＳ法）によるプロテオーム解析技術によってがん転移関連タンパク質を同定する。 In FIG. 1, “LM-BxPC-3”, which is a highly metastatic cell line of pancreatic cancer, is created using NOG mice from “BxPC-3”, which is a cell line derived from pancreatic cancer. BxPC-3 and LM-BxPC-3 are cultured and protein extraction is performed. From the extracted protein samples BxPC-3 and LM-BxPC-3, a cancer metastasis-related protein is identified by a proteome analysis technique by an isotope labeling method (NBS method) using 2-nitrobenzenesulfenyl chloride (NBSCl).
例えば、大腸がん由来細胞株である「ＳＷ４８」についても、同様にして、ＮＯＧマウスを用いて大腸がん高転移細胞株である「ＬＭ−ＳＷ４８」を作成する。その他のがん細胞株についても、がん高転移細胞株を作成することができる。 For example, for “SW48” which is a colon cancer-derived cell line, “LM-SW48” which is a colon cancer high metastasis cell line is similarly prepared using NOG mice. A cancer highly metastatic cell line can be created for other cancer cell lines.
ＮＢＳ法において、２種類のタンパク質試料（元の親細胞株及びがん高転移細胞株、図１においては、元の親細胞株ＢｘＰＣ−３及びがん高転移細胞株ＬＭ−ＢｘＰＣ−３）のうち一方を重い試薬（２−ニトロ［13Ｃ6］ベンゼンスルフェニルクロリド）で修飾し、他方を軽い試薬（２−ニトロ［12Ｃ6］ベンゼンスルフェニルクロリド）で修飾し、得られたＮＢＳ修飾タンパク質試料双方を互いに混合し、トリプシン消化など当業者による適切な処理を行い、質量分析装置でペプチド量の違いを測定する。このことにより、前記２種類のタンパク質試料におけるタンパク質含有量の相対的な差を調べることができる。ＮＢＳ法は、Rapid Commun. Mass Spectrom., 2003, 17, 1642-1650及び国際公報第２００４／００２９５０号パンフレットなどにも記載されている。 In the NBS method, two types of protein samples (original parent cell line and cancer high metastasis cell line, in FIG. 1, the original parent cell line BxPC-3 and cancer high metastasis cell line LM-BxPC-3) One of them is modified with a heavy reagent (2-nitro [ 13 C 6 ] benzenesulfenyl chloride), the other is modified with a light reagent (2-nitro [ 12 C 6 ] benzenesulfenyl chloride), and the obtained NBS modification Both protein samples are mixed with each other, subjected to an appropriate treatment by a person skilled in the art such as trypsin digestion, and the difference in peptide amount is measured with a mass spectrometer. This makes it possible to examine the relative difference in protein content between the two types of protein samples. The NBS method is also described in Rapid Commun. Mass Spectrom., 2003, 17, 1642-1650 and International Publication No. 2004/002950 pamphlet.
図２を参照して、抽出されたタンパク質試料として、細胞抽出液 (ＴＬ：Total lysate) そのものを用いて解析することができる。さらに、細胞抽出液 (Total lysate)を遠心処理して、上清と沈殿とに分離する。上清をリン酸タンパク質濃縮カラム処理に付して、リン酸タンパク質濃縮画分（Ｐｉ）を得て、リン酸タンパク質濃縮画分（Ｐｉ）をタンパク質試料として、ＮＢＳ法によるプロテオーム解析を行う。また、沈殿を尿素による可溶化処理に付して、尿素可溶化画分（Ｕｒ）を得て、尿素可溶化画分（Ｕｒ）をタンパク質試料として、ＮＢＳ法によるプロテオーム解析を行う。 With reference to FIG. 2, it can analyze using a cell extract (TL: Totallysate) itself as an extracted protein sample. Further, the cell extract (Total lysate) is centrifuged to separate the supernatant and the precipitate. The supernatant is subjected to a phosphoprotein concentration column treatment to obtain a phosphoprotein enriched fraction (Pi), and a proteome analysis by the NBS method is performed using the phosphoprotein enriched fraction (Pi) as a protein sample. The precipitate is subjected to a solubilization treatment with urea to obtain a urea-solubilized fraction (Ur), and the urea-solubilized fraction (Ur) is used as a protein sample for proteomic analysis by the NBS method.
これらのタンパク質は、当業者によって選択されるあらゆるタンパク質精製技術によって単離及び精製することができる。例えば、イオン交換、アフィニティ、及びサイズ排除カラムクロマトグラフィーなどのクロマトグラフィー、遠心分離、溶解度差、及び電気泳動などを含む技術を用いることができる。 These proteins can be isolated and purified by any protein purification technique selected by one skilled in the art. For example, techniques including chromatography such as ion exchange, affinity, and size exclusion column chromatography, centrifugation, differential solubility, and electrophoresis can be used.
このような手法により、本発明者らは、”Zinc finger protein 185”、”Cell surface antigen 4F2 heavy chain”、”Isocitrate dehydrogenase 1”、”Interleukin enhancer-binding factor 3”、”Transcription factor IIF, alpha subunit”、”Torsin-1A-interacting protein 1”、”Activated RNA polymerase II transcriptional coactivator p15”、及び “Clathrin heavy chain 1” からなる群から選ばれる少なくとも１種のタンパク質を含むがん転移マーカーを見出した。 By such a technique, the present inventors have made use of “Zinc finger protein 185”, “Cell surface antigen 4F2 heavy chain”, “Isocitrate dehydrogenase 1”, “Interleukin enhancer-binding factor 3”, “Transcription factor IIF, alpha subunit. A cancer metastasis marker comprising at least one protein selected from the group consisting of “,“ Torsin-1A-interacting protein 1 ”,“ Activated RNA polymerase II transcriptional coactivator p15 ”, and“ Clathrin heavy chain 1 ”was found.
実施例１（表１）において詳しく示されるが、全細胞抽出液 (ＴＬ)、リン酸タンパク質濃縮画分（Ｐｉ）及び尿素可溶化画分（Ｕｒ）の各試料について、がん高転移細胞株よりも親細胞株について発現亢進しているがん転移マーカーもあるし、逆に、親細胞株よりもがん高転移細胞株について発現亢進しているがん転移マーカーもある。 As shown in detail in Example 1 (Table 1), cancer high metastasis cell lines were obtained for each sample of whole cell extract (TL), phosphoprotein enriched fraction (Pi) and urea solubilized fraction (Ur). There are also cancer metastasis markers whose expression is enhanced in the parent cell line, and conversely, there are cancer metastasis markers whose expression is enhanced in the cancer high metastasis cell line than in the parent cell line.
本発明のがん転移マーカーは、例えば以下の用途で用いられる。組織中のマーカーの発現レベルを測定することによって、例えば、がん患者の原発がんの他の部位への転移（例えば、肝転移）診断や予後診断を行うために用いられうる。また、ＰＥＴなどの画像診断において用いられるプローブの標的として用いられうる。或いは、がん転移の治療標的として用いられうる。 The cancer metastasis marker of the present invention is used in the following applications, for example. By measuring the expression level of a marker in a tissue, it can be used, for example, for diagnosis or prognosis of metastasis (for example, liver metastasis) to other parts of a primary cancer of a cancer patient. Moreover, it can be used as a target of a probe used in diagnostic imaging such as PET. Alternatively, it can be used as a therapeutic target for cancer metastasis.
［ガン転移マーカーの分析方法］
本発明は、上記タンパク質群から選ばれる少なくとも１種のタンパク質をがん転移マーカーとして使用することにより試料を分析する方法を提供する。本発明のがん転移マーカーの分析方法は、生体試料中のがん転移マーカーの発現レベルを取得する工程と、前記がん転移マーカーの基準レベルに基づき、前記発現レベルの高低によって評価を行う工程とを含む。
[Method of analyzing cancer metastasis marker]
The present invention provides a method for analyzing a sample by using at least one protein selected from the above protein group as a cancer metastasis marker. The method for analyzing a cancer metastasis marker according to the present invention includes a step of obtaining an expression level of a cancer metastasis marker in a biological sample, and a step of evaluating the expression level based on the reference level of the cancer metastasis marker. Including.
すなわち、本発明の方法において、上述のタンパク質は、がん患者体内で基準レベルに比べて発現量が亢進又は抑制するマーカーとして用いられる。基準レベルは、がん患者由来のがん性試料における上記がん転移マーカーレベルの対照となるレベルでありうる。例えば、転移がないがん患者由来のがん性試料などにおける上記がん転移マーカーレベルが挙げられる。 That is, in the method of the present invention, the above-mentioned protein is used as a marker whose expression level is increased or suppressed compared to a reference level in a cancer patient. The reference level can be a level that serves as a control for the cancer metastasis marker level in a cancerous sample derived from a cancer patient. For example, the cancer metastasis marker level in a cancerous sample derived from a cancer patient without metastasis can be mentioned.
本発明の方法においては、分析に供されるべき試料を用意し、当該試料から、上記タンパク質群から選ばれる少なくとも１種のがん転移マーカーの発現レベルを取得する。当該がん転移マーカーの基準レベルに基づいて、取得した発現レベルの高低に関する評価を行う。がん転移マーカーの種類に応じて、発現レベルが基準レベルより高い又は低いとの評価をもって、当該試料が由来する個体ががん転移に罹患している可能性が高い、あるいは、今後がん転移に罹患する可能性が高い、ことの指標とすることができる。 In the method of the present invention, a sample to be subjected to analysis is prepared, and the expression level of at least one cancer metastasis marker selected from the protein group is obtained from the sample. Based on the reference level of the cancer metastasis marker, an evaluation is performed regarding the level of the acquired expression level. Depending on the type of cancer metastasis marker, the expression level is higher or lower than the reference level, and the individual from which the sample is derived is likely to have cancer metastasis or cancer metastasis in the future. It can be used as an indicator of a high possibility of suffering from the disease.
分析に供されるべき試料としては、がん転移の罹患あるいは将来的罹患を識別すべき対象となる個人に由来する生体試料でありうる。 The sample to be subjected to the analysis can be a biological sample derived from an individual who is a subject to be identified for cancer metastasis or future morbidity.
生体試料は、生体から採取した試料であることが好ましい。生体から採取した試料は、培養細胞株を除く意である。生体から採取した試料は、生体内での生命現象が直接反映されているものである。 The biological sample is preferably a sample collected from a living body. A sample collected from a living body is intended to exclude cultured cell lines. A sample collected from a living body directly reflects a life phenomenon in the living body.
分析に供されるべき試料としては、特に限定されない。例えば、細胞や組織、体液、及び組織抽出物などが挙げられる。細胞や組織には、組織生検材料などが含まれる。組織生検材料には、生検組織が含まれ、検査・手術などで採取されうる。特に、手術で切除された組織が挙げられる。それら組織は、組織切片、組織破砕物又は後述の組織抽出物の態様で提供されてよい。当該組織は、原発巣に由来するものでありうる。例えば、大腸由来組織としては、粘膜上皮、粘膜固有層、粘膜筋板、粘膜下層、固有筋層、及び漿膜などが挙げられる。体液としては、血液、尿、及び体分泌物などが含まれる。血液としては、全血、血漿、血清などが含まれる。組織抽出物とは、当業者に公知の方法によってホモジネート又は可溶化された組織をいう。 The sample to be subjected to analysis is not particularly limited. Examples include cells, tissues, body fluids, and tissue extracts. Cells and tissues include tissue biopsy materials. The tissue biopsy material includes a biopsy tissue and can be collected by examination or surgery. In particular, a tissue excised by surgery may be mentioned. These tissues may be provided in the form of tissue sections, tissue fragments or tissue extracts described below. The tissue can be derived from a primary focus. For example, examples of the large intestine-derived tissue include mucosal epithelium, lamina propria, mucosal muscle plate, submucosa, lamina propria and serosa. Body fluids include blood, urine, and body secretions. The blood includes whole blood, plasma, serum and the like. Tissue extract refers to tissue homogenated or solubilized by methods known to those skilled in the art.
上記タンパク質の発現レベルの取得は、例えば、生体特異的親和性に基づく検査によって行われうる。生体特異的親和性に基づく検査は当業者に良く知られた方法であり、特に限定されないが、イムノアッセイが好ましい。具体的には、ウエスタンブロット、ラジオイムノアッセイ、ＥＬＩＳＡ、サンドイッチイムノアッセイ、免疫沈降法、沈降反応、免疫拡散法、免疫凝集測定、補体結合反応分析、免疫放射定量法、蛍光イムノアッセイ、プロテインＡイムノアッセイなどの、競合及び非競合アッセイ系を含むイムノアッセイが含まれる。イムノアッセイにおいては、個体の試料中のマーカーに結合する抗体の存在を検出する。具体的には、アッセイ試料中において、測定すべきマーカータンパク質及び当該タンパク質の抗体からなる免疫複合体を形成しうる条件のもと、試料を当該抗体に接触させることによって行われる。より具体的なイムノアッセイプロトコルは、当業者であれば容易に選択することができる。 Acquisition of the expression level of the protein can be performed, for example, by a test based on biospecific affinity. The test based on biospecific affinity is a method well known to those skilled in the art, and is not particularly limited, but an immunoassay is preferable. Specifically, Western blot, radioimmunoassay, ELISA, sandwich immunoassay, immunoprecipitation method, precipitation reaction, immunodiffusion method, immunoagglutination measurement, complement binding reaction analysis, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay, etc. Immunoassays, including competitive and non-competitive assay systems are included. In an immunoassay, the presence of an antibody that binds to a marker in a sample of an individual is detected. Specifically, it is carried out by bringing the sample into contact with the antibody under the conditions that can form an immune complex consisting of the marker protein to be measured and the antibody of the protein in the assay sample. More specific immunoassay protocols can be easily selected by those skilled in the art.
或いは、上記タンパク質の発現レベルの取得は、上記の生体特異的親和性に基づく方法以外のタンパク質定量法によって測定することもできる。例えば、既に述べたＮＢＳ法は、定量性に優れた方法である。この場合、上記タンパク質を既知レベルで調製した試料や、正常試料などの適当な試料を対照試料として、分析対象試料との間における前記タンパク質の存在量の差を調べることによって、測定を行うことができる。 Or acquisition of the expression level of the said protein can also be measured by protein quantification methods other than the method based on said biospecific affinity. For example, the NBS method already described is an excellent method for quantitativeness. In this case, the measurement can be performed by examining the difference in the abundance of the protein from the sample to be analyzed, using a sample prepared with a known level of the protein or an appropriate sample such as a normal sample as a control sample. it can.
或いは、ＮＢＳ法でなくとも、質量分析計を用いた種々の分析手法に基づいて、当該タンパク質或いはそれに由来するペプチドの分析から、発現レベルの取得や評価を行っても良い。 Alternatively, the expression level may be acquired and evaluated from analysis of the protein or a peptide derived therefrom based on various analysis methods using a mass spectrometer, instead of the NBS method.
本発明の方法が実施される態様の一例として、以下が挙げられる。
本態様においては、分析に供される試料として、手術などで切除を行ったがん組織（より具体的には原発巣組織）の切片が用いられる。この組織は免疫組織化学染色に供され、免疫組織化学染色によって生じた染色の強度、並びに染色の分布（単位面積当たりの染色割合）を併せてマーカーの発現レベルの指標とする。基準レベルは、転移なし（例えば、肝転移なし）のがん患者（例えば、膵臓がん患者、大腸がん患者）におけるがん部位の染色強度、並びに染色の分布（単位面積当たりの染色割合）を考慮したものでありうる。例えば、染色強度が基準レベルより強い領域が、分析に供された組織の３０％以上の範囲に分布していることをもって、取得した発現レベルが基準レベルに基づいて高いと評価することができる。あるいは、例えば、染色強度が基準レベルより弱い領域が、分析に供された組織の３０％以上の範囲に分布していることをもって、取得した発現レベルが基準レベルに基づいて低いと評価することができる。がん転移マーカーの種類に応じて、適宜の評価用閾値を定めるとよい。
或いは、上記例においては、発現レベルの取得及び評価をマスイメージング法に基づいて行っても良い。
The following is mentioned as an example of the aspect by which the method of this invention is implemented.
In this embodiment, as a sample to be analyzed, a section of cancer tissue (more specifically, primary lesion tissue) excised by surgery or the like is used. This tissue is subjected to immunohistochemical staining, and the intensity of staining produced by immunohistochemical staining and the distribution of staining (staining ratio per unit area) are used as indicators of the expression level of the marker. The reference level is the intensity of staining at the cancer site in cancer patients (eg, pancreatic cancer patients, colon cancer patients) without metastasis (eg, liver metastasis), and the distribution of staining (staining rate per unit area). Can be considered. For example, it can be evaluated that the acquired expression level is high based on the reference level because the region where the staining intensity is stronger than the reference level is distributed in a range of 30% or more of the tissue subjected to analysis. Alternatively, for example, when the region where the staining intensity is weaker than the reference level is distributed in a range of 30% or more of the tissue subjected to analysis, it is evaluated that the acquired expression level is low based on the reference level. it can. An appropriate threshold for evaluation may be determined according to the type of cancer metastasis marker.
Alternatively, in the above example, acquisition and evaluation of the expression level may be performed based on a mass imaging method.
本発明の方法が実施される態様の他の一例として、以下が挙げられる。
本態様においては、分析に供される試料として、生検組織などを破砕又は抽出に供して調製されたものが用いられる。この試料はマーカータンパク質の濃度測定に供され、得られた測定値が、マーカーの発現レベルの情報となる。基準レベルには、例えば、別の試料において得られた当該マーカーの測定値や、当該マーカーに固有の閾値が含まれる。この態様においては、測定値が基準レベルと比較され、測定値が基準レベルを超えていることをもって、取得した発現レベルが基準レベルに基づいて高いと評価することができる。あるいは、測定値が基準レベルよりも小さいことをもって、取得した発現レベルが基準レベルに基づいて低いと評価することができる。
The following is mentioned as another example of the aspect by which the method of this invention is implemented.
In this embodiment, a sample prepared by crushing or extracting a biopsy tissue or the like is used as a sample to be analyzed. This sample is subjected to marker protein concentration measurement, and the obtained measurement value becomes information on the expression level of the marker. The reference level includes, for example, a measurement value of the marker obtained in another sample and a threshold unique to the marker. In this aspect, the measured value is compared with the reference level, and when the measured value exceeds the reference level, it can be evaluated that the acquired expression level is high based on the reference level. Or it can be evaluated that the acquired expression level is low based on a reference level because a measured value is smaller than a reference level.
或いは、上記他の一例においては、試料が採取された血液試料であってよい。その場合、前記血液試料は、生体内に戻されることなく破棄される。 Alternatively, in another example described above, the blood sample may be a collected blood sample. In that case, the blood sample is discarded without being returned to the living body.
本発明の方法において、本発明のがん転移マーカーは単独で用いられてもよいし、他のいかなるマーカーと組み合わせて用いられてもよい。従って、本発明の方法は、本発明のがん転移マーカーの発現レベル情報の取得と同様に他のマーカーの発現レベル情報を取得することを含んでいてよい。 In the method of the present invention, the cancer metastasis marker of the present invention may be used alone or in combination with any other marker. Therefore, the method of the present invention may include obtaining the expression level information of other markers as well as obtaining the expression level information of the cancer metastasis marker of the present invention.
［治療標的分子及び創薬標的分子］
本発明のがん転移マーカーは、がん（例えば、膵臓がん、大腸がん）における転移性の他のがん（例えば、転移性肝がん）の治療のための治療標的分子、又はがんの転移抑制や転移性がんの治療のための創薬標的分子となりうる。
[Therapeutic target molecule and drug target molecule]
The cancer metastasis marker of the present invention is a therapeutic target molecule for the treatment of other metastatic cancers (eg, metastatic liver cancer) in cancer (eg, pancreatic cancer, colon cancer), or It can be a drug discovery target molecule for inhibiting metastasis of cancer and treating metastatic cancer.
例えば、大腸がん肝転移の治療には、肝転移した大腸がん細胞を死滅させることを含む。大腸がんの肝転移抑制には、大腸がん細胞の肝転移抑制、又は転移性肝がん細胞を死滅させることを含む。 For example, treatment of colon cancer liver metastases includes killing colon cancer cells that have metastasized to the liver. Inhibiting liver metastasis of colorectal cancer includes inhibiting liver metastasis of colon cancer cells or killing metastatic liver cancer cells.
本発明のがん転移マーカーを治療標的分子又は創薬標的分子とすることにより、以下の医薬候補分子が提供されうる。 By using the cancer metastasis marker of the present invention as a therapeutic target molecule or a drug discovery target molecule, the following drug candidate molecules can be provided.
当該医薬候補分子の一態様は、治療標的分子又は創薬標的分子に対する特異性を有するものである。より具体的には、治療標的分子又は創薬標的分子に免疫特異的に結合する抗体を含むものが挙げられる。抗体は、ポリクローナル抗体、モノクローナル抗体、及び、分子生物学的技術により調製した抗体を含む。ここで抗体とは、広く免疫特異的に結合する物質であればよく、抗体フラグメントや抗体融合タンパク質も用いられてよい。いずれの場合も、抗体の調製は、当業者に良く知られた方法によって行われる。また、抗体は、創薬分野における抗体工学技術によって通常なされる分子改変及び修飾が施されてよい。上記医薬候補分子は、原発性（例えば、大腸）がん細胞に対して供給されることによって、転移の抑制及び転移性がん細胞の死滅、又は成長を抑制する反応を惹起しうる。 One embodiment of the drug candidate molecule has specificity for a therapeutic target molecule or a drug discovery target molecule. More specifically, those containing an antibody that immunospecifically binds to a therapeutic target molecule or a drug discovery target molecule can be mentioned. Antibodies include polyclonal antibodies, monoclonal antibodies, and antibodies prepared by molecular biological techniques. Here, the antibody may be any substance that binds widely immunospecifically, and an antibody fragment or an antibody fusion protein may also be used. In either case, antibody preparation is carried out by methods well known to those skilled in the art. In addition, the antibody may be subjected to molecular alteration and modification usually performed by antibody engineering techniques in the field of drug discovery. When the drug candidate molecule is supplied to primary (for example, large intestine) cancer cells, it can elicit reactions that suppress metastasis and kill or metastasize metastatic cancer cells.
医薬候補分子の他の一態様は、本発明のがん転移の治療標的分子を含むものである。上記医薬候補分子は、免疫刺激量で原発性がん細胞に対して供給されることによって、転移（肝転移）の抑制および転移性がん細胞（転移性肝がん細胞）の死滅、又は成長を抑制する免疫応答を惹起しうる。ここで、免疫刺激量とは、がんの処理のために所望する免疫反応を惹起することが可能な抗原の量をいい、当業者によって良く知られた方法で決定されるものである。この形態によると、いわゆるがんワクチン療法として知られている、当業者に良く知られた方法を用いてがんの処理が行われる。 Another embodiment of the drug candidate molecule includes the cancer metastasis therapeutic target molecule of the present invention. The drug candidate molecule is supplied to primary cancer cells in an immunostimulatory amount, thereby suppressing metastasis (liver metastasis) and killing or growing metastatic cancer cells (metastatic liver cancer cells). Can elicit an immune response that suppresses Here, the immunostimulatory amount refers to the amount of an antigen capable of eliciting a desired immune response for the treatment of cancer, and is determined by a method well known by those skilled in the art. According to this form, cancer treatment is performed using a method well known to those skilled in the art, known as so-called cancer vaccine therapy.
上記医薬候補分子は、自らを有効成分とし、薬剤として許容される希釈剤、担体、賦形剤などをさらに含むことによって薬剤組成物となりうる。上記薬剤組成物は、がん転移の治療や原発性がんの転移抑制に用いられる潜在的な治療剤として特定されうる。或いは、上記薬剤組成物は、がん転移の治療や原発性がんの転移（肝転移）抑制に用いられる治療剤として用いられうる。 The drug candidate molecule can be a pharmaceutical composition by containing itself as an active ingredient and further containing a pharmaceutically acceptable diluent, carrier, excipient and the like. The pharmaceutical composition can be identified as a potential therapeutic agent used to treat cancer metastasis or to suppress metastasis of primary cancer. Or the said pharmaceutical composition can be used as a therapeutic agent used for the treatment of cancer metastasis, and primary cancer metastasis (liver metastasis) suppression.
以下に実施例を示し、本発明を具体的に説明するが、本発明は下記の実施例に制限されるものではない。 EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.
［実施例１］
上記非特許文献２に従って、ＮＯＧ mouseを用いて作成した「がん転移モデル系」を利用することで、ヒトの「がん高転移細胞株」を樹立した。この「がん高転移細胞株」と元の「親細胞株」とを用いて、２−ニトロベンゼンスルフェニルクロリド（ＮＢＳ）試薬を用いた解析システムによる定量プロテオーム解析を行った。その結果、がん転移に関連する多数の蛋白質（以下、「がん転移関連蛋白質」と記載）を同定することができた。図１及び図２には、膵臓がん細胞株である「ＢｘＰＣ−３」の場合を例として、ＮＢＳ法解析システムを利用した定量プロテオーム解析の手順を示すフロー図が示されている。
[Example 1]
According to Non-Patent Document 2, a “cancer metastasis cell line” created using NOG mouse was used to establish a human “cancer metastasis cell line”. Using this "cancer metastasis cell line" and the original "parent cell line", quantitative proteome analysis was performed by an analysis system using 2-nitrobenzenesulfenyl chloride (NBS) reagent. As a result, a large number of proteins related to cancer metastasis (hereinafter referred to as “cancer metastasis-related proteins”) could be identified. FIG. 1 and FIG. 2 are flowcharts showing the procedure of quantitative proteome analysis using the NBS method analysis system, taking the case of “BxPC-3” which is a pancreatic cancer cell line as an example.
膵臓がん由来細胞株である「ＢｘＰＣ−３」、並びに大腸がん由来細胞株である「ＳＷ４８」の２つの系について、上記定量プロテオーム解析を行ったところ、１１種類の蛋白質が両方の系で共通の挙動を示す、すなわち、どちらの系においても高転移株でのタンパク質発現量が亢進しているか、或いは、どちらの系においても高転移株でのタンパク質発現量が低下している「がん転移関連蛋白質」として見出された（表１）。 When the above quantitative proteome analysis was performed on two systems, “BxPC-3”, which is a pancreatic cancer-derived cell line, and “SW48”, which is a colon cancer-derived cell line, 11 types of proteins were found in both systems. It shows a common behavior, that is, the protein expression level in the high metastasis strain is increased in either system, or the protein expression level in the high metastasis strain is decreased in either system. It was found as "metastasis-related protein" (Table 1).
培養細胞を凍結融解させて細胞を破砕し、遠心後の上清を2D clean up kit（GEヘルスケア）を用いて精製したものを「全細胞抽出液（ＴＬ）」、遠心後の上清をQIAGEN社のPhosphoprotein purification kitを用いてリン酸タンパク質の濃縮を行ったものを「リン酸タンパク質濃縮画分（Ｐｉ）」、遠心後の沈殿を10M 尿素溶液に懸濁してもう一度遠心した後の上清を「尿素可溶化画分（Ｕｒ）」として、それぞれ定量プロテオーム解析に用いた。 The cultured cells are frozen and thawed to disrupt the cells, and the supernatant after centrifugation is purified using a 2D clean up kit (GE Healthcare). The whole cell extract (TL) is obtained, and the supernatant after centrifugation is obtained. Phosphoprotein purification kit using QIAGEN Phosphoprotein purification kit is the “phosphoric acid protein enriched fraction (Pi)”. The supernatant after centrifugation is suspended in 10M urea solution and centrifuged again Was used as a “urea-solubilized fraction (Ur)” for quantitative proteome analysis.
これら全細胞抽出液（ＴＬ）、リン酸タンパク質濃縮画分（Ｐｉ）、および尿素可溶化画分（Ｕｒ）の各蛋白質試料をそれぞれＮＢＳ試薬により処理を行い、蛋白質の定量解析を行った。ＮＢＳ試薬処理に関しては、13C NBS Stable Isotope Labeling Kit-N(島津製作所)の推奨プロトコルに従い、安定同位体標識を行った。得られたそれぞれのラベル化蛋白質を混合し、キットの推奨プロトコルに従い、脱塩・還元・アルキル化、およびトリプシン消化を行った。次に、同キットのプロトコルに従い、ラベル化ペプチドを濃縮した。濃縮したラベル化ペプチドに関しては、引き続いてC18カラムを用いたμHPLCによる分離を行い、MSプレートに塗布した。このようにして準備したサンプルに関して質量分析装置AXIMA-CFR plus（島津製作所製）を用いて測定し、各蛋白質の相対定量解析を行った。 Each protein sample of the whole cell extract (TL), phosphate protein-enriched fraction (Pi), and urea-solubilized fraction (Ur) was treated with an NBS reagent, and the protein was quantitatively analyzed. Regarding NBS reagent treatment, stable isotope labeling was performed according to the recommended protocol of 13 C NBS Stable Isotope Labeling Kit-N (Shimadzu Corporation). Each of the obtained labeled proteins was mixed, and desalted, reduced, alkylated, and trypsin digested according to the recommended protocol of the kit. Next, the labeled peptide was concentrated according to the protocol of the kit. Concentrated labeled peptides were subsequently separated by μHPLC using a C18 column and applied to MS plates. The sample thus prepared was measured using a mass spectrometer AXIMA-CFR plus (manufactured by Shimadzu Corporation), and a relative quantitative analysis of each protein was performed.
m/z値が900以上で、なおかつ相対比率が大きいもの（一方を100としたときに他方が150以上となるもの）を目安に測定対象ピークを選択して、質量分析装置AXIMA-QIT（島津製作所製）を用いてMS/MS解析による蛋白質同定を行った。 Select the peak to be measured based on an m / z value of 900 or more and a large relative ratio (one is 100 and the other is 150 or more). Mass spectrometer AXIMA-QIT (Shimadzu) (Manufactured by Seisakusho) was used for protein identification by MS / MS analysis.
表１には、ＢｘＰＣ−３及びＳＷ４８の２つの細胞系について共通の挙動を示す１１種類の蛋白質が「がん転移関連蛋白質」として示されている。表１における略号は以下の意味を表す。なお、Alpha-actinin 1 / Alpha-actinin 4は、マスコット検索において同定されたアミノ酸配列”Identified Sequence; LASDLLEWIR”を含む元のタンパク質が2種類あることを示している。 In Table 1, eleven types of proteins exhibiting a common behavior for the two cell lines BxPC-3 and SW48 are shown as “cancer metastasis-related proteins”. The abbreviations in Table 1 have the following meanings. Alpha-actinin 1 / alpha-actinin 4 indicates that there are two types of original proteins including the amino acid sequence “Identified Sequence; LASDLLEWIR” identified in the mascot search.
Ｐｒｅｐ：タンパク質試料
ＴＬ：全細胞抽出液(Total Lysate)
Ｐｉ：リン酸タンパク質濃縮画分
Ｕｒ：尿素可溶化画分
Prep: Protein sample TL: Whole cell extract (Total Lysate)
Pi: Phosphate protein concentrated fraction Ur: Urea-solubilized fraction
Ｒａｔｉｏ：タンパク質の存在（発現）比率（複数回実験を行った場合は、その平均値）
ＬＭ：「がん高転移細胞株」における発現が、元の「親細胞株」における発現を上回っていたことを示す。この場合、比率の値は、元の「親細胞株」における発現（ピーク面積／強度）を１００（基準）としたときの「がん高転移細胞株」における発現の量（ピーク面積／強度）を表す。
Ｂｘ又はＳＷ：元の「親細胞株」における発現が、「がん高転移細胞株」における発現を上回っていたことを示す。この場合、比率の値は、「がん高転移細胞株」における発現（ピーク面積／強度）を１００（基準）としたときの元の「親細胞株」における発現の量（ピーク面積／強度）を表す。
なお、∞は、「がん高転移細胞株」におけるピーク又は元の「親細胞株」におけるピークのうちの一方が検出限界以下であったことを示す。
例えば、「ＬＭ∞」は、「がん高転移細胞株」における発現が、元の「親細胞株」における発現を上回っており、元の「親細胞株」におけるピークが検出限界以下であったことを示す。
Ratio: Protein presence (expression) ratio (when multiple experiments are performed, the average value)
LM: Indicates that the expression in the “cancer metastasis cell line” exceeded the expression in the original “parent cell line”. In this case, the ratio value is the amount of expression (peak area / intensity) in the “cancer metastasis cell line” when the expression (peak area / intensity) in the original “parent cell line” is 100 (reference). Represents.
Bx or SW: Indicates that the expression in the original “parent cell line” was higher than the expression in the “cancer metastasis cell line”. In this case, the value of the ratio is the amount of expression (peak area / intensity) in the original “parent cell line” when the expression (peak area / intensity) in the “cancer highly metastatic cell line” is 100 (reference). Represents.
In addition, ∞ indicates that one of the peak in the “cancer high metastasis cell line” or the peak in the original “parent cell line” was below the detection limit.
For example, in “LM∞”, the expression in the “cancer high metastasis cell line” exceeds the expression in the original “parent cell line”, and the peak in the original “parent cell line” was below the detection limit. It shows that.
１１種類の「がん転移関連蛋白質」のうち、例えば、アクチニン4（actinin-4）は、その過剰発現が細胞の運動性を誘導し、転移を促進することが、培養細胞や動物を用いた実験で明らかとなっているうえ、臨床的にもその発現と転移との関連が報告されている（非特許文献４）。同様に、Glucosidase II (β-glucosidase)については、薬剤による阻害実験等でその活性と転移との関連が報告されている（非特許文献５）。beta-N-acetylhexosaminidase （beta-hexosaminidase / N-acetyl-beta- glucosaminidase）については、血液中等の酵素活性と転移の程度に相関があることが知られている（非特許文献６、７）。これらの報告において、本実施例にて、２つの転移モデル系で「がん転移関連蛋白質」として同定されたもののいくつかは、がん転移との相関やがん転移マーカーとしての利用可能性が示されている。このことから、残る８種類の蛋白質についてもがん転移の原因因子である、或いは、がん転移のマーカーとして使える可能性が十分に考えられたため、更に以下の解析を進めた。 Among 11 types of “cancer metastasis-related proteins”, for example, actinin-4 (actinin-4) used cultured cells and animals that overexpression induces cell motility and promotes metastasis. In addition to being clarified by experiments, clinically, the relationship between its expression and metastasis has been reported (Non-patent Document 4). Similarly, regarding Glucosidase II (β-glucosidase), a relationship between its activity and metastasis has been reported in an inhibition experiment with a drug or the like (Non-patent Document 5). Regarding beta-N-acetylhexosaminidase (beta-hexosaminidase / N-acetyl-beta-glucosaminidase), it is known that there is a correlation between enzyme activity in blood and the degree of transfer (Non-patent Documents 6 and 7). In these reports, some of those identified as "cancer metastasis-related proteins" in the two metastasis model systems in this example are correlated with cancer metastasis and can be used as cancer metastasis markers. It is shown. From these facts, the remaining 8 types of proteins were considered to be causative factors of cancer metastasis or could be used as markers for cancer metastasis. Therefore, the following analysis was further advanced.
［実施例２］
次に、大腸がん患者１１０例の切除組織断片について、免疫組織化学染色を行った。本手法の免疫組織化学染色実験に用いた大腸がん患者の臨床情報を表２に示す。
[Example 2]
Next, immunohistochemical staining was performed on excised tissue fragments of 110 colon cancer patients. Table 2 shows the clinical information of colorectal cancer patients used in the immunohistochemical staining experiment of this method.
染色のターゲットとして、上記実施例１で発見したがん転移関連蛋白質のうちの一つ、Zinc finger protein 185（以下、ZNF185と記載）を用いた。図３は、ZNF185による大腸がん組織の免疫組織化学染色における典型的な写真であり、図３（Ａ）は染色が見られなかった場合(Negative)、図３（Ｂ）は染色が見られた場合(Positive)の典型的写真である。 One of the cancer metastasis-related proteins discovered in Example 1 above, Zinc finger protein 185 (hereinafter referred to as ZNF185) was used as a staining target. FIG. 3 is a typical photograph in immunohistochemical staining of colon cancer tissue with ZNF185. FIG. 3 (A) shows no staining (Negative), and FIG. 3 (B) shows staining. This is a typical photograph of (Positive).
ZNF185に対する特異抗体を用いた染色の有無を、リンパ節転移、肝転移、静脈侵襲といった患者の臨床情報と照らし合わせて医学統計解析を行い、臨床での病態とマーカー染色の関連を精査した。 We performed a medical statistical analysis of the presence or absence of staining with a specific antibody against ZNF185 against clinical information of patients such as lymph node metastasis, liver metastasis, and venous invasion, and examined the relationship between clinical pathology and marker staining.
表３に、ZNF185蛋白質の発現と肝転移との相関を示す。「肝転移あり」は術後に発見された例を含んでいる。Fisher正確確率検定を行った場合、片側（one-sided）検定の場合はp=0.039、両側（two-sided）検定の場合はp=0.029となり、どちらの条件による検定でも「ZNF185染色陽性」と「肝転移有り」との関連が有意であることがわかった。次に、ZNF185染色陽性により肝転移の存在を予測する感度と特異度を示した。ここでは染色の程度を考慮せず、陽性・陰性の２種類のみに分類したが、染色強度を数値等で判定し、なおかつ適当な閾値で分類することで、感度と特異度の割合を検査の狙いに応じて調整することも可能と考える。 Table 3 shows the correlation between the expression of ZNF185 protein and liver metastasis. “With liver metastases” includes cases discovered postoperatively. When the Fisher exact test is performed, p = 0.039 for the one-sided test and p = 0.029 for the two-sided test. The relationship with “with liver metastasis” was found to be significant. Next, the sensitivity and specificity for predicting the presence of liver metastasis by positive ZNF185 staining were shown. Here, the degree of staining is not considered, and only two types, positive and negative, are classified. However, by determining the staining intensity numerically and classifying it with an appropriate threshold, the ratio between sensitivity and specificity can be determined. I think that it is possible to adjust according to the aim.
表４に、ZNF185蛋白質の発現とその他臨床情報との相関を示す。性別、部位2、静脈侵襲、リンパ管侵襲、浸潤様式については、Fisherの正確検定による p値を、部位1、深達度、組織型（分化度）、Stageについてはカイ二乗（Pearson）検定による p値を示す。 Table 4 shows the correlation between the expression of ZNF185 protein and other clinical information. For gender, site 2, venous invasion, lymphatic invasion, and invasion mode, Fisher's exact p-value is used. For site 1, depth of penetration, tissue type (differentiation), and stage is chi-square (Pearson) test. p value is shown.
これらの結果、ZNF185による染色強度の高低が、肝転移と有意に相関することがわかった。つまり、ZNF185蛋白質が高発現していた患者では、低発現の患者に比べて肝臓へ転移した人の割合が有意に多いことがわかった。 As a result, it was found that the intensity of staining with ZNF185 significantly correlated with liver metastasis. In other words, it was found that the proportion of those who metastasized to the liver was significantly higher in patients with high expression of ZNF185 protein than in patients with low expression.
また、Kaplan-Meier法により、患者の生存曲線とマーカー染色の有無との相関について解析を行った。図４は、患者の生存日数（日）に対する累積生存を示すKaplan-Meier法による生存曲線である。その結果、統計的な有意差はみられなかったものの、ZNF185蛋白質が高発現していた患者の方が、生存率は低かった。 In addition, the Kaplan-Meier method was used to analyze the correlation between patient survival curves and the presence or absence of marker staining. FIG. 4 is a survival curve according to the Kaplan-Meier method showing cumulative survival with respect to the survival days (days) of patients. As a result, although there was no statistically significant difference, the survival rate was lower in patients with high expression of ZNF185 protein.
Cox比例ハザードモデルによる生存分析を行った。その結果を表５に示す。 Survival analysis by Cox proportional hazard model was performed. The results are shown in Table 5.
表５より、肝転移や病理深達度と同様、ZNF185の発現強度の高低が予後に影響を与える独立因子であることがわかった。 From Table 5, it was found that the level of expression intensity of ZNF185 is an independent factor affecting the prognosis, as is the case with liver metastasis and pathological depth.
Claims (5)
The biological sample includes a crushed tissue, the expression level is a measured concentration of the cancer metastasis protein marker detected in the sample, and the reference level is a reference concentration of the cancer metastasis protein marker The method of claim 2 , wherein
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