JP2008011728A - がん転移の判定方法及び装置 - Google Patents
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Abstract
【解決手段】がん細胞の転移が疑われるリンパ節から調製された検出用試料中の腫瘍マーカーmRNAを定量する工程、及び前記mRNAの定量結果に基づいて前記リンパ節における転移巣の大きさを判定する工程を含むがん転移の判定方法を提供する。
【選択図】なし
Description
R1−R2−(CH2CH2O)n−H
(ここで、R1は炭素数10〜22のアルキル基、アルケニル基、アルキニル基、イソオクチル基;R2は−O−又は−(C6H6)−O−;nは8〜120の整数)
で表されるポリオキシエチレン系非イオン性界面活性剤が好適であり、例えばポリオキシエチレンラウリルエーテル、ポリオキシエチレンセチルエーテル、ポリオキシエチレンオレイルエーテル、ポリオキシエチレンミリスチリルエーテル、ポリオキシエチレンステアリルエーテル、ポリオキシエチレンノニルフェニルエーテル、ポリオキシエチレンイソオクチルフェニルエーテルなどが挙げられる。具体的には、Brij35(ポリオキシエチレン(35)ラウリルエーテル)などが好適である。緩衝液中の界面活性剤の濃度は0.1〜6%(v/v)が好ましく、より好ましくは1〜5%(v/v)である。
(1)免疫組織化学染色
乳がん患者から切除したリンパ節組織11個と、大腸がん患者から切除したリンパ節7個を用いて、免疫組織化学染色による転移巣サイズの測定を行った。
上記で切り取った切片に隣接する切片(厚さ約10μm)を切り取り、RNeasy Mini Kit(キアゲン社)を用いてRNA抽出を行い、RNA試料を調製した。このRNA試料を用い、以下の組成の反応液を調製してTaqMan法によってCK19mRNAのコピー数を算出した。なお、TaqMan法による測定は、TaqMan One-step RT-PCR Master Mix及びPrism 7700 Realtime PCR system(何れもアプライドバイオシステムズ社)を用いて添付の使用説明書に従って行われた。
精製水 20.10μL
RNA試料 2μL
TaqMan 2X Universal PCR Master Mix 25μL
40X MultiScribe and RNase Inhibitor Mix 1.25μL
100μM フォワードプライマ 0.15μL
100μM リバースプライマ 0.15μL
7.4pmol/μL(終濃度200nm) TaqManプローブ 1.35μL
フォワードプライマ:5’-CAGATCGAAGGCCTGAAGGA-3’(配列番号1)
リバースプライマ:5’-CTTGGCCCCTCAGCGTACT-3’(配列番号2)
TaqManプローブ:5’-GCCTACCTGAAGAAGAACCATGAGGAGGAA-3’(配列番号3)
乳頭腺管癌に由来するがん細胞が転移したリンパ節1個から、一定の間隔を置いて厚さ約10μmの切片を35枚切り出した。この35枚の切片に対して実施例1(1)と同様にして免疫組織化学染色を行い、がん細胞の数(個/セクション)を計数した。
免疫組織化学染色に用いた切片をmRNAの定量に用いることはできないため、この切片に隣接する切片(厚さ約10μL)のmRNA発現量(コピー/セクション)を実施例1と同様にして測定し、この定量結果を免疫組織化学染色によるがん細胞数と対応させた。mRNA発現量とがん細胞数の関係を図9に示す。
実施例2と同様にして、がん細胞計数に供した所定の切片におけるmRNA発現量を、隣接する切片のmRNA発現量とし、mRNA発現量とがん細胞数の関係を図10に示す。
がん転移が認められないリンパ節11個(陰性検体)を用い、実施例1(2)と同様にして検出用試料を作製してそれぞれの検体におけるCK19mRNA発現量(バックグラウンドの値)を測定した。
また、表1に示す乳がんのデータを基に、各切片において転移巣が一辺1mmの立方体(体積1mm3)であると仮定した場合のCK19mRNA発現量と、一辺2mmの立方体(体積8mm3)であると仮定したときのCK19mRNA発現量とを算出した。
さらに、転移巣が1mm3であった場合のCK19mRNA発現量及び8mm3であった場合のCK19mRNA発現量に、バックグラウンド値である陰性検体のCK19mRNA発現量をそれぞれ加算した。即ち、これらの値は、転移巣を有さないリンパ節一個のCK19mRNA発現量(バックグラウンド)、1mm3の転移巣を有するリンパ節一個のCK19mRNA発現量の推定値、及び8mm3の転移巣を有するリンパ節一個のCK19mRNA発現量の推定値である。これらの値を図11に示す。
本実施例では、第1の閾値は、バックグラウンドの最高値である3.0E+06コピー以上に設定することができる。
乳がん患者から採取されたリンパ節64個(陰性検体42個、陽性検体22個)、及び大腸がん患者から採取されたリンパ節69個(陰性検体33個、陽性検体36個)(直径約6mm/個)に、それぞれ、pH3.4の緩衝液(200mMグリシン−HCl、5% Brij35(ポリオキシエチレン(35)ラウリルエーテル、シグマ社製)及び20% DMSO(和光純薬)を含む)4mLを添加し、ブレンダーでホモジナイズした。10000×gで1分間遠心し、上清を採取した。この上清を上記緩衝液で10倍に希釈し、検出用試料(lysate)2μLを採取した。
また、図12においてCK19mRNAの発現量(コピー/μL・lysate)が第2の閾値以上であった検体は乳がんのマクロメタスタシスを含むことが予想され、第1の閾値以上第2の閾値未満であった検体は乳がんのマイクロメタスタシスを含むことが予想される。
また、図13においてCK19mRNAの発現量(コピー/μL・lysate)が第2の閾値以上であった検体は乳がんのマクロメタスタシスを含むことが予想され、第1の閾値以上第2の閾値未満であった検体は乳がんのマイクロメタスタシスを含むことが予想される。
Claims (10)
- がん細胞の転移が疑われるリンパ節から調製された検出用試料中の腫瘍マーカーmRNAを定量する工程、及び
前記mRNAの定量結果に基づいて前記リンパ節における転移巣の大きさを判定する工程を含む、がん転移の判定方法。 - がん細胞の転移が疑われるリンパ節から調製された検出用試料中の腫瘍マーカーmRNAを定量する工程、及び
前記mRNAの定量結果に基づいて前記リンパ節における転移巣の大きさ又は転移巣に含まれるがん細胞数を定量する工程を含む、がん転移の判定方法。 - 前記転移巣の大きさ又は転移巣に含まれるがん細胞数を定量する工程において、
前記mRNAの定量結果を、前記mRNAの定量結果に対応する第1の閾値と比較し、前記mRNAの定量結果が前記第1の閾値未満である場合は前記リンパ節ががん転移陰性であると判定し、前記定量結果が前記第1の閾値以上である場合は、前記転移巣を定量する、請求項2記載の方法。 - がん細胞の転移が疑われるリンパ節から調製された検出用試料中の腫瘍マーカーmRNAを定量する工程、及び
前記mRNAの定量結果を、前記定量結果に対応する第1の閾値及び前記第1の閾値より高値である第2の閾値と比較することにより、前記リンパ節ががん転移陰性、がん転移弱陽性、及びがん転移強陽性の何れであるかを判定する工程を含む、がん転移の判定方法。 - 前記mRNAの定量結果が、前記検出用試料中の前記mRNAの絶対量である、請求項1〜4の何れかに記載の方法。
- 前記腫瘍マーカーがサイトケラチン19である、請求項1〜5の何れかに記載の方法。
- 前記第1の閾値が50〜3000コピー/μL・lysateに設定される、請求項3又は4記載の方法。
- 前記第1の閾値が200〜300コピー/μL・lysateに設定される、請求項3又は4記載の方法。
- 前記第2の閾値が2000〜20000コピー/μL・lysateに設定される、請求項4記載の方法。
- がん細胞の転移が疑われるリンパ節から調製された検出用試料中の腫瘍マーカーmRNAを定量する定量手段、及び
前記mRNAの定量結果を、前記定量結果に対応する第1の閾値及び前記第1の閾値より高値である第2の閾値と比較することにより、前記リンパ節ががん転移陰性、がん転移弱陽性、及びがん転移強陽性の何れであるかを判定する判定手段を含む、がん転移の判定装置。
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