胃癌リンパ節微小転移の蛍光即時PCR法による検出と臨床的意義の研究

荧光瞬时PCR法检测胃癌淋巴结微转移及临床意义研究

• 批准号: 13770689
• 负责人: 久保田 啓介
• 金额: $ 0.96万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13770689/
• 关键词: 微小転移; リンパ節; 胃癌; real-time RT-PCR; CEA; CK20; PCR; 免疫染色

本研究では、real-time RT-PCR法による胃癌リンパ節微小転移の検出能を検討し、その検出能をH-E、免疫組織染色法と比較した。方法:対象は21例の胃癌患者のリンパ節392個と非癌リンパ節1個。全393個のリンパ節を3群に分類:病理陽性群71個、病理陰性群304個、対照群18個(非癌リンパ節1個とm癌の遠隔リンパ節17個)。全てのリンパ節は、半分をH-Eと免疫組織染色に用い、残る半分をreal-time RT-PCRに用いた。AGPC変法にてRNAを抽出し、続いてcDNAを作成。LyghtCyclerを用い、CEA・CK20を標的としたreal-time RT-PCRを行った。蛍光プローブはハイブリダイゼーションプローブを用いた。GAPDHのreal-time RT-PCRも行った。AE1/AE3、CEA、CK20の免疫染色を行い、染色強度を評価した。平均16ヶ月追跡。結果:CEA mRNAの平均値は病理陽性群、病理陰性群、対照群の各々で16,000、21、0.6。非補正CEA値を使用。カットオフ5.0で、1個を除く病理陽性群全てと、病理陰性群中26個が陽性。CK20 mRNAでは、病理陰性群中16個が陽性で、病理陽性群中13個が陰性。CEA、CK20の一方が陽性のリンパ節を陽性と判定する解析法で、病理陽性群全ては陽性、病理陰性群中30個が陽性。病理陽性群全てはAE1/AE3免疫組織染色陽性で、病理陰性群中の11個が陽性。陽性率はH-E、免疫組織染色、複合real-time RT-PCRで各々18.0%、20.9%、25.8%。病理陰性・複合real-time RT-PCR陽性の30個中9個で免疫組織染色で微小転移が確認された。AE1/AE3陽性の11人中、CEAとCK20では各々3人と5人が染色されなかった。mRNA測定値と染色強度はCEAでもCK20でも有意に相関した。早期癌9症例では病理陰性の2個がreal-time RT-PCR陽性、si患者の1例では病理学的に1群転移のみ認めたがreal-time RT-PCRで2群転移を認めた。この3例は現在まで無再発。1例は肝転移再発死亡し、他に4例の腹膜播種再発・肝転移再発を認めた。討論:本研究は胃癌リンパ節微小転移の検出にreal-time RT-PCR法を用いた最初のものである。本法は定量的で迅速であるため臨床応用性が広い。カットオフを定めることにより偽陽性を排除し、複合real-time RT-PCR法にて偽陰性を排除しうる。微小転移が通常のリンパ流領域に存在したこと、少数個の集塊から成る転移巣であったことが、今回予後がよかった理由と考えた。Real-time RT-PCR法は胃癌リンパ節微小転移の検出において鋭敏な検出法である。

This study is based on real-time RT-PCR method, gastric cancer micro-transfer method, H-E, and immunohistochemical staining method. Methods: We selected 21 gastric cancer patients with 392 nodules and 1 non-cancerous nodule. A total of 393 のリンパパを3 groups were classified: 71 pathologically positive groups, 304 pathologically negative groups, and 18 radiological groups (1 non-cancerous リンパパを and 17 cancerous remote のンパパに). The whole section is used for H-E immunohistochemistry staining, and the remaining half section is used for real-time RT-PCR. The AGPC method is used to extract RNA and create cDNA. LyghtCycler is used and CEA・CK20 is the standard for real-time RT-PCR.荍光プローブはハイブリダイゼーションプローブを用いた. GAPDH's real-time RT-PCR function. The results of immunostaining and staining intensity for AE1/AE3, CEA, and CK20 were evaluated. Average tracking is 16 months. Results: The average values ​​of CEA mRNA were 16,000, 21, and 0.6 for the pathologically positive group, the pathologically negative group, and the negative group, respectively. Non-corrected CEA values ​​are used. There were 5.0 カットオフで, 1 をout of all pathologically positive groups, and 26 がpositives in the pathologically negative group. CK20 mRNA was not detected, 16 of the pathologically negative group were positive, and 13 of the pathologically positive group were negative. One of the positive cases of CEA and CK20 is determined by the analysis method, all cases in the pathologically positive group are positive, and 30 cases in the pathologically negative group are positive. All of the pathologically positive group were positive for AE1/AE3 immunohistochemical staining, and 11 of the pathologically negative group were positive. The positive rates of H-E, immune tissue staining, and composite real-time RT-PCR were 18.0%, 20.9%, and 25.8% respectively. Nine out of 30 pathologically negative and composite real-time RT-PCR positive cases were not confirmed by immunohistological staining and microscopic changes. Among 11 people who were positive for AE1/AE3, 3 and 5 people each were stained with CEA and CK20, respectively. The mRNA measurement value and staining intensity were significantly correlated with CEA and CK20. Nine cases of early-stage cancer were pathologically negative, and 2 were real-time RT-PCR positive, and 1 patient was pathologically confirmed by real-time RT-PCR. None of the 3 cases have happened again now. One patient died due to liver metastasis and recurrence, and 4 other cases were diagnosed after peritoneal inoculation and recurrence of liver disease. Discussion: In this study, the real-time RT-PCR method of gastric cancer was first used to detect microscopic changes in gastric cancer. This method has quantitative, rapid and clinical applicability. The カットオフを定めることによりfalse positives are excluded, and the composite real-time RT-PCR method is used to exclude false negatives. There is a small amount of normal flow in the field, a small number of lumps, a small number of pieces, and a reason why I will do it now. The real-time RT-PCR method is used for gastric cancer, and it is a very sensitive method for detecting microscopic changes in gastric cancer.

项目成果

Keisuke Kubota, et al. (10 authors): "Quantitative detection of micrometastasis in the lymph nodes of gastric cancer patients with real-time RT-PCR : A comparative study with immuno-histochemistry"International Journal of Cancer. Vol.104(in press). (2003)

久保田圭介等人。