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One-Step Nucleic Acid Amplification法によるリンパ節検査は、肺腫瘍の病期分類をより正確にすることができるか？
Can the One-Step Nucleic Acid Amplification method of lymph nodes examination make the staging of pulmonary tumours more precise?
PMID: 30441990
抄録
序論:
本論文の目的は、原発性（非小細胞）肺腫瘍と二次性（大腸癌の転移）肺腫瘍の場合、ヘマトキシリン・エオシン染色による標準的な病理組織学的検査、抗サイトケラチン19抗体による免疫組織化学的検査、およびOne-Step Nucleic Acid Amplification法に基づく検査を用いて、肺門リンパ節および縦隔リンパ節の微小転移を検出する感度を比較することである。
INTRODUCTION: The aim of this article is to compare the sensitivity of detecting micrometastases in hilar and mediastinal lymph nodes in case of primary (non-small cell) and secondary (metastases of colorectal carcinoma) pulmonary tumours using standard histopathological examination with haematoxylin-eosin staining, immunohistochemistry examination with Anti-Cytokeratin 19 antibody and examination based on the One-Step Nucleic Acid Amplification method.
方法:
原発性非小細胞肺癌および肺転移大腸癌の根治的手術治療の際に、2015 年から 2017 年までに本研究に登録された 100 名の患者の胸膜リンパ節および縦隔リンパ節を標準的な分類に基づいて抽出した。その後、これらのリンパ節を縦軸に沿って4つの同一部位に分割し、左側の第1部と第3部はOne-Step Nucleic Acid Amplification法による検査、第2部と第4部は病理組織学的検査を行った。組織学的検査による結節の各部位の評価は、まずヘマトキシリン・エオシン染色を行い、その後、抗サイトケラチン19抗体を用いた免疫組織化学的検査を行うという標準的な方法で行った。ワンステップ核酸増幅法は、シスメックス社（神戸市）から提供されたキットを用いて、サイトケラチン１９のｍＲＮＡ（メッセンジャーリボ核酸）を等温増幅と結合した逆転写により検出する方法である。
METHOD: During radical surgical treatment of primary non-small cell lung carcinoma and pulmonary metastases of colorectal carcinoma, hilar and mediastinal lymph nodes of 100 patients enrolled in the study in the period from 2015 to 2017 were extracted based on a standard classification. These lymph nodes were subsequently divided along the longitudinal axis into 4 identical parts where part one and three on the left were intended for examination based on the One-Step Nucleic Acid Amplification method, whereas parts two and four were subjected to histopathological examination. In evaluating the respective parts of the nodes by histological examination, the nodes were first examined by a standard procedure that involves haematoxylin-eosin staining, followed by immunohistochemistry examination with Anti-Cytokeratin 19 antibody. The One-Step Nucleic Acid Amplification method was performed in the kit supplied by Sysmex (Kobe, Japan) and is based on the detection of cytokeratin 19 mRNA (messenger ribonucleic acid) by reverse transcription coupled with isothermal amplification.
結果:
本研究に登録された患者の合計1,426例のリンパ節を抽出し、上記の方法で検査を行った。78人の患者（78％）では、ヘマトキシリン・エオシン染色、抗サイトケラチン19を用いた免疫組織化学、ワンステップ核酸増幅法を用いても同じ結果が得られた。他の方法を用いずにOne-Step Nucleic Acid Amplification法を用いたリンパ節内の微小転移は16例（16％）で証明された。One-Step Nucleic Acid Amplification法が陰性であったのに対し、ヘマトキシリン・エオシン染色による検査や抗サイトケラチン19による免疫組織化学検査が陽性であったのは3例（3％）のみであった。抗サイトケラチン19抗体を用いた免疫組織化学で得られた結果は、ヘマトキシリン・エオシン染色で得られた結果と実質的に同じであった（97％）。
RESULTS: A total of 1,426 lymph nodes of the patients enrolled in the study were extracted and examined using the above mentioned methodology. In 78 patients (78%), identical results were obtained using haematoxylin-eosin staining, immunohistochemistry with Anti-Cytokeratin 19 and One-Step Nucleic Acid Amplification. Micrometastases in the lymph nodes using the One-Step Nucleic Acid Amplification method in the absence of the other methods were proven in 16 patients (16%). Only in 3 cases (3%), the examination by haematoxylin-eosin staining, or immunohistochemistry with Anti-Cytokeratin 19, was positive while One-Step Nucleic Acid Amplification was negative. The results obtained by immunohistochemistry with Anti-Cytokeratin 19 antibody were practically the same as those obtained by haematoxylin-eosin staining (97%).
結論:
CONCLUSION: The results of the study have demonstrated a higher percentage of metastases detected in hilar and mediastinal lymph nodes if the One-Step Nucleic Acid Amplification method of examination was used compared to haematoxylin-eosin staining and immunohistochemistry with Anti-Cytokeratin 19 antibody (upstaging in 16%). This shows that the examination of lymph nodes using the One-Step Nucleic Acid Amplification method can have a certain potential to make the pulmonary tumours staging more accurate. On the other hand, immunohistochemistry with Anti-Cytokeratin 19 antibody seems to be not so useful. However, it is necessary to prove this hypothesis in follow-up studies, or where applicable, in a larger cohort of patients. Another task is to ascertain, by careful patient monitoring, the influence of the micrometastases detected in their lymph nodes using the One-Step Nucleic Acid Amplification method on these patients' follow-up. Key words: lung cancer - lymph nodes - H&E - IHC CK19 - OSNA assay.