JP4004385B2 - Cancer detection method by double staining - Google Patents
Cancer detection method by double staining Download PDFInfo
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- JP4004385B2 JP4004385B2 JP2002324433A JP2002324433A JP4004385B2 JP 4004385 B2 JP4004385 B2 JP 4004385B2 JP 2002324433 A JP2002324433 A JP 2002324433A JP 2002324433 A JP2002324433 A JP 2002324433A JP 4004385 B2 JP4004385 B2 JP 4004385B2
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- cancer
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【0001】
【発明の属する技術分野】
この出願の発明は、二重染色法によるガン検出方法に関するものである。さらに詳しくは、この出願の発明は、近年注目されるガン細胞増殖阻害剤であるハーセプチン等の治療対象症例(HER−2陽性)の選択に有用な、形態学的シグナルと遺伝子レベルのシグナルとが明瞭に可視画像として判別可能とされる、高精度なガン検出方法に関するものである。
【0002】
【従来の技術】
HER−2タンパク質を産生する遺伝子、すなわち、HER2/neu遺伝子はがん遺伝子のひとつとして注目されており、1986年にその配列が決定されている。この遺伝子はチロシンキナーゼ活性をもつ受容体型のタンパクで分子量185kDaの膜貫通型タンパクをコードしており、細胞外のシグナルを細胞内に伝達し細胞増殖を促進する機能を担っている。そして、乳癌や卵巣癌・胃癌において高頻度に増幅または過剰発現していることが知られている。このためHER2/neu遺伝子の増幅あるいはタンパクの過剰発現は、癌の早期再発や予後不良と相関し、乳癌や卵巣癌・胃癌などの臨床経過を予測する上で非常に有用である。特に乳癌においては、全患者の25〜30%においてHER−2タンパクの過剰発現がみられ、こういった患者では転移をきたしやすく、進行が早いため生存率も低いことがわかっている。
【0003】
そこで、新規なガン治療剤のスクリーニング等においては、一般病理検査の対象とされた細胞がHER−2タンパク質を過剰発現しているかどうかを迅速に判定し、しかも対象とされる細胞を高精度に検出・診断することが重要な課題になっている。
【0004】
このような状況において、免疫染色法による判定方法が開発され、また、螢光マーカーの付与による観察との比較方法が提案されている(文献1および2)。
【0005】
【文献】
【表1】
【0006】
【発明が解決しようとする課題】
しかしながら、従来の方法においては、免疫染色によるHER−2発現状況と予後および治療効果の予測との間にはばらつきが認められたり、一方乳癌の10%前後で遺伝子の増幅なしにタンパクの発現のみが過剰となる例が認められるとの報告もある。
【0007】
このため、ガン診断のための方法としては精度が低いのが実情であった。そしてこのことは、従来の免疫組織染色法と螢光マーカー付与による比較観察においては、形態的細胞膜シグナルと核内染色体シグナルとの判別ができないという状況をともなっていた。
【0008】
この出願の発明は、以上のとおりの従来技術の問題点を解消し、HER−2陽性、つまりHER−2タンパク質発現の対象症例について、高精度でガン検出することを可能とする新しい技術手段を提供することを課題としている。
【0009】
【課題を解決するための手段】
この出願の発明は、上記の課題を解決するものとして、第1には、次の手順によりHER−2陽性の対象例についてガン組織を検出することを特徴とする二重染色法によるガン検出方法を提供する。
【0010】
(I)病理標本を免疫染色して細胞膜をDAB発色させた後、
【0011】
(II)(II)細胞染色体に螢光マーカーを付与し、蛍光レーザー顕微鏡で染色体を観察する。次いで、
【0012】
(III)前記細胞染色体にアルカリホスファターゼ標識抗FITC抗体を反応させてニューフクシン発色させて、ガン細胞の細胞染色体および形状を可視化する。
【0013】
そして、第2には、細胞染色体への螢光マーカーの付与は、マイクロウェーブ間欠照射による螢光In situハイブリダイゼーションとして行うことを特徴とする上記の二重染色法によるガン検出方法を提供する。
【0014】
【発明の実施の形態】
上記のとおりの特徴を有するこの出願の発明は、HER−2陽性の治療対象症例の選択に有効であって、免疫組織化学的方法(IHC)と、蛍光In situハイブリダイゼーション(FISH、暗視野で蛍光レーザー顕微鏡で観察)並びに明視野での可視化(CISH)との2重染色を用いることにより、乳癌、卵巣癌、胃がん、肺癌等のガン組織の高精度検出を可能としている。
【0015】
その手順は▲1▼免疫染色(IHC)▲2▼染色体への螢光マーカー付与(FISH)、▲3▼ニューフクシン発色(CISH)である。
【0016】
すなわち、患者から患部組織を採取し常法に従い作成されたパラフィン包埋薄切り病理標本を、たとえば、先ず米DAKO社の手法
(たとえば、http://www.nisiq.net/〜dakoj/her/senshoku.htmlを参照することができる)
に準じて処理しHER−2陽性の患者特有に見られるガン細胞表層に過剰生産されたHER−2タンパク質をDAB染色により褐色に発色(可視化)させてその発色強度を判定する(IHCステップ)。次いで細胞染色体に螢光マーカーを付ける(FISH法:この段階で暗視野下螢光レーザー顕微鏡観察すると染色体は緑色点として観測され、ガン細胞は染色体数異常(正常細胞に比べて数が多い)が見られる。しかしFISH法だけでは可視領域の光学顕微鏡では観察できないため、ガン細胞部分と正常細胞部分とを形状で同時に区別できない。そこで螢光観察後、さらにCISH処理を組み合わせニューフクシン発色させることにより、ガン細胞の癌特異蛋白(HER-2)を茶褐色に、染色体動原体を紫色の点として発色させて可視化観測する。
【0017】
たとえばその手順が上記のとおりのこの出願の発明による形態学的シグナルと遺伝子シグナルのシグナルとが組合された特有の可視画像はガン細胞の高精度判定において極めて有用である。
【0018】
そして、この出願の発明においては、従来二重染色を行うと、形態的シグナルと核内染色体シグナルが重なって十分な染め分けができないとされてきたが、最初に免疫染色(IHC)を行い、細胞膜をDAB染色し、さらにFISH処理時のハイブリダイゼーション工程におけるマイクロウェーブ(MW)間欠照射処理条件を最適化することにより、形態学的シグナルと核内染色体増幅シグナルとが重なることなく染め分けることが可能とされている。
【0019】
マイクロウェーブ(MW)間欠照射について、たとえば、出力コントロールが約150W〜400Wの範囲において可変とされ、MW照射中に温度が急激に上昇する場合には出力を下げ、温度の上昇が好ましくないときは出力を挙げることで照射中にも可能とされるようにする。ハイブリダイゼーションにおけるMWの間欠照射の温度は40〜45℃、出力は4/300W、3秒照射/2秒停止の繰返しの操作条件がたとえば好適に採用される。
【0020】
従来法では、IHC染色とFISHを別に行い、各々を観測比較したのに対して、この出願の発明では同一細胞でタンパク発現と遺伝子増幅を同時に観察できることから、より的確で信頼性の高い判定が可能となる。
【0021】
そこで以下により具体的にこの出願の発明の実施の形態について説明する。
【0022】
<A> HER−2/neu.IHC
以下の手順とする。
【0023】
【表2】
【0024】
添付した図面の図1は、HER−2タンパク質をDAB染色により褐色に発色(可視化)させた状態を示した白黒の写真である。
【0025】
<B> FISH
以下の手順とする。
【0026】
【表3】
【0027】
暗視野下螢光レーザー顕微鏡観察すると染色体は緑色点として観察され、ガン細胞には染色体異常が見られる。図2は、この観察像を白黒写真として例示したものである。
【0028】
<C> CISH
以下の手順とする。
【0029】
【表4】
【0030】
FISHだけでは染色体シグナルが可視光線の光学顕微鏡では観察できないがCISH後には、染色体動原体が紫色の点として発色され、可視化観察することができる。
【0031】
図3は、この観察像を白黒写真として例示したものである。
【0032】
【発明の効果】
以上のとおりのこの出願の発明によって、HER−2陽性の対象例選択に有用な、形態学的シグナルと遺伝子レベルのシグナルとが明瞭に可視画像として判別可能とされる、高精度なガン検出方法が提供される。
【0033】
なお、米国およびヨーロッパにおけるHER2検査ガイドラインでは通常
1)FIRST LineとしてIHC法を実施し、結果3+の場合治療薬の投与を決定する。
【0034】
2)IHC法の結果2+の場合、再検およびFISH法での確認を推奨する。とされている。この出願の発明法は、乳癌をはじめとするガン診断法として大いに注目を集めるものと期待される。
【図面の簡単な説明】
【図1】IHC後の観察像を例示した写真である。
【図2】FISH後の観察像を例示した写真である。
【図3】CISH後の観察像を例示した写真である。[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a cancer detection method by a double staining method. More specifically, the invention of this application relates to a morphological signal and a gene level signal that are useful for selecting a case to be treated (HER-2 positive) such as Herceptin, which is a cancer cell growth inhibitor that has recently attracted attention. The present invention relates to a highly accurate gun detection method that can be clearly distinguished as a visible image.
[0002]
[Prior art]
A gene that produces HER-2 protein, that is, the HER2 / neu gene has attracted attention as one of oncogenes, and its sequence was determined in 1986. This gene is a receptor type protein having tyrosine kinase activity and encodes a transmembrane protein having a molecular weight of 185 kDa, and has a function of transmitting an extracellular signal into the cell and promoting cell proliferation. And it is known that it is frequently amplified or overexpressed in breast cancer, ovarian cancer and gastric cancer. For this reason, HER2 / neu gene amplification or protein overexpression correlates with early recurrence of cancer and poor prognosis, and is very useful in predicting clinical course of breast cancer, ovarian cancer, gastric cancer, and the like. In particular, in breast cancer, HER-2 protein is overexpressed in 25 to 30% of all patients, and it is known that such patients are prone to metastasis and progress rapidly so that the survival rate is low.
[0003]
Therefore, in screening for a novel cancer therapeutic agent or the like, it is quickly determined whether or not the cells targeted for the general pathological examination overexpress the HER-2 protein, and the targeted cells are highly accurate. Detection and diagnosis is an important issue.
[0004]
In such a situation, a determination method using an immunostaining method has been developed, and a comparison method with observation by applying a fluorescent marker has been proposed (References 1 and 2).
[0005]
[Literature]
[Table 1]
[0006]
[Problems to be solved by the invention]
However, in the conventional method, there is a variation between the HER-2 expression status by immunostaining and the prediction of prognosis and therapeutic effect, whereas only about 10% of breast cancers without protein amplification. There are reports that there are examples of excessive amounts of.
[0007]
For this reason, the actual situation is that the accuracy of cancer diagnosis is low. This was accompanied by a situation in which morphological cell membrane signals and nuclear chromosome signals could not be discriminated in the conventional observation by immunohistochemical staining and fluorescence marker application.
[0008]
The invention of this application solves the problems of the prior art as described above, and provides a new technical means that enables cancer detection with high accuracy in a HER-2 positive, that is, a target case of HER-2 protein expression. The issue is to provide.
[0009]
[Means for Solving the Problems]
The invention of this application is to solve the above-mentioned problems. First, a cancer detection method by double staining , wherein cancer tissue is detected for a HER-2 positive target example by the following procedure. I will provide a.
[0010]
(I) After immunostaining the pathological specimen to develop DAB color on the cell membrane ,
[0011]
(II) (II) A fluorescent marker is added to the cell chromosome, and the chromosome is observed with a fluorescence laser microscope. Then
[0012]
(III) The cell chromosome is reacted with an alkaline phosphatase-labeled anti-FITC antibody to develop neufuxin color, and the cell chromosome and shape of the cancer cell are visualized.
[0013]
Then, in the second, giving a fluorescent marker to the cells chromosomes, that Kyosu Hisage cancer detection method of the double staining method which is characterized in that as a fluorescence In situ hybridization with microwave intermittent irradiation .
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The invention of this application having the characteristics as described above is effective in selecting a HER-2 positive treatment target case, and includes immunohistochemical method (IHC), fluorescence in situ hybridization (FISH, dark field). By using double staining with observation with a fluorescent laser microscope and visualization in bright field (CISH), cancer tissue such as breast cancer, ovarian cancer, stomach cancer, lung cancer and the like can be detected with high accuracy.
[0015]
The procedures are (1) immunostaining (IHC), (2) fluorescent marker addition (FISH) to the chromosome, and (3) new fuchsin color development (CISH).
[0016]
That is, a paraffin-embedded sliced pathological specimen prepared by extracting a diseased part tissue from a patient according to a conventional method, for example, a method of the US DAKO company (for example, http://www.nisiq.net/~dakoj/her/senshoku) .html can be referenced)
The HER-2 protein, which is processed according to HER-2 and is overproduced on the surface of the cancer cell, which is peculiar to HER-2 positive patients, is colored (visualized) brown by DAB staining to determine the color intensity (IHC step). Next, a fluorescent marker is attached to the cell chromosome (FISH method: When observed under a fluorescent laser microscope in the dark field at this stage, the chromosome is observed as a green dot, and the cancer cell has an abnormal number of chromosomes (more than normal cells). However, since the FISH method alone cannot be observed with an optical microscope in the visible region, the cancer cell part and the normal cell part cannot be distinguished simultaneously by shape. The tumor cell-specific protein (HER-2) of the cancer cell is colored brown and the chromosome centromere is colored as a purple dot and visualized.
[0017]
For example, a specific visible image combining a morphological signal and a gene signal according to the invention of this application as described above is very useful in high-accuracy determination of cancer cells.
[0018]
In the invention of this application, it has been said that, when conventional double staining is performed, morphological signals and nuclear chromosomal signals overlap so that sufficient dyeing cannot be performed. First, immunostaining (IHC) is performed, By DAB staining and optimization of microwave (MW) intermittent irradiation treatment conditions in the hybridization process during FISH treatment, it is possible to separate morphological signals and nuclear chromosome amplification signals without overlapping. It is said that.
[0019]
For microwave (MW) intermittent irradiation, for example, when the output control is variable in a range of about 150 W to 400 W, and the temperature is suddenly increased during MW irradiation, the output is decreased, and the temperature increase is not preferable. By raising the output, it will be possible even during irradiation. In the hybridization, the temperature of intermittent irradiation of MW is 40 to 45 ° C., the output is 4/300 W, and the operation condition of repetition of 3 seconds irradiation / 2 seconds stop is preferably adopted, for example.
[0020]
In the conventional method, IHC staining and FISH were performed separately, and each was observed and compared. In the invention of this application, protein expression and gene amplification can be observed simultaneously in the same cell, so a more accurate and reliable determination can be made. It becomes possible.
[0021]
Therefore, an embodiment of the invention of this application will be specifically described below.
[0022]
<A> HER-2 / neu. IHC
The following procedure is used.
[0023]
[Table 2]
[0024]
FIG. 1 of the accompanying drawings is a black-and-white photograph showing a state where the HER-2 protein is colored brown (visualized) by DAB staining.
[0025]
<B> FISH
The following procedure is used.
[0026]
[Table 3]
[0027]
When observed with a fluorescent laser microscope in the dark field, the chromosomes are observed as green spots, and the chromosome abnormalities are seen in the cancer cells. FIG. 2 illustrates this observed image as a black and white photograph.
[0028]
<C> CISH
The following procedure is used.
[0029]
[Table 4]
[0030]
Chromosome signals cannot be observed with a visible light optical microscope with FISH alone, but after CISH, the chromosomal centromere is colored as a purple dot and can be visualized.
[0031]
FIG. 3 illustrates this observation image as a black and white photograph.
[0032]
【The invention's effect】
According to the invention of this application as described above, a highly accurate cancer detection method that is useful for selecting a HER-2 positive target example and that can clearly distinguish a morphological signal and a gene level signal as a visible image. Is provided.
[0033]
In addition, in the HER2 test guideline in the United States and Europe, usually 1) IHC method is implemented as FIRST Line, and in the case of result 3+, the administration of the therapeutic agent is determined.
[0034]
2) If the result of the IHC method is 2+, re-examination and confirmation by the FISH method are recommended. It is said that. The invention method of this application is expected to attract a great deal of attention as a cancer diagnostic method including breast cancer.
[Brief description of the drawings]
FIG. 1 is a photograph illustrating an observation image after IHC.
FIG. 2 is a photograph illustrating an observation image after FISH.
FIG. 3 is a photograph illustrating an observation image after CISH.
Claims (2)
(I)病理標本を免疫染色して細胞膜をDAB発色させた後、
(II)細胞染色体に螢光マーカーを付与し、蛍光レーザー顕微鏡で染色体を観察する。次いで、
(III)前記細胞染色体にアルカリホスファターゼ標識抗FITC抗体を反応させてニューフクシン発色させて、ガン細胞の細胞染色体および形状を可視化する。A cancer detection method by a double staining method , wherein cancer tissue is detected for a HER-2 positive target example by the following procedure.
(I) After immunostaining the pathological specimen to develop DAB color on the cell membrane ,
(II) A fluorescent marker is added to the cell chromosome, and the chromosome is observed with a fluorescence laser microscope. Then
(III) The cell chromosome is reacted with an alkaline phosphatase-labeled anti-FITC antibody to develop neufuxin color, and the cell chromosome and shape of the cancer cell are visualized.
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US20150322498A1 (en) | 2012-08-09 | 2015-11-12 | Japanese Foundation For Cancer Research | Method for labelling of cleavage of nucleic acid sequence |
US10352859B2 (en) * | 2013-09-26 | 2019-07-16 | Konica Minolta, Inc. | Method for determining quantity of biological material in tissue section |
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CN113358615B (en) * | 2021-06-01 | 2023-05-12 | 张慧敏 | Application of Melan and sodium fluorescein double staining method in living cell imaging |
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