cbcbba100μm100μm100μm100μmSerial sections of tumor tissueSerial sections of tumor tissueSerial sections of normal tissueSerial sections of normal tissue372cases for EBV assay372cases for EBV assay363EBV-negative cases for MSI analysis363EBV-negative cases for MSI analysisH&E stained slides for DNA extractionH&E stained slides for DNA extractionH&E stained slides for DNA extractionH&E stained slides for DNA extractionIntratumor: inside of the tumor borderIntratumor: inside of the tumor borderInvasive margin:along the tumor border, where half of the field of view (20x mag) contains tumor tissueInvasive margin:along the tumor border, where half of the field of view (20x mag) contains tumor tissueH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EPan-cytokeratinPan-cytokeratinH&EH&ECKCKCKCKx20x20x20x20100μm100μm100μm100μmH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&EH&ETracing tumor borderTracing tumor borderSelecting hot spots of the Selecting hot spots of the most lymphocyte-rich area most lymphocyte-rich area H&EH&EH&EH&E21 cases lacking paired tumor/normal DNA21 cases lacking paired tumor/normal DNA9 cases with EBV-positive tumor9 cases with EBV-positive tumorSerial sectionsSerial sectionsx20x20x20x20IHCIHCIHCIHCIHCIHCH&EH&EIHCIHCH&EH&ECD8CD8Selected 3 hot spots at the intratumor areaSelected 3 hot spots at the intratumor areaSelected 3 hot spots at the invasive marginSelected 3 hot spots at the invasive marginOne hot spotOne hot spotCD8CD8One hot spotOne hot spotCD8CD8100μm100μm100μm100μm100μm100μm100μm100μmUnstained slides for immunohistochemistryUnstained slides for immunohistochemistrySerial sectionsSerial sectionsx20x20x20x20IHCIHCIHCIHCIHCIHCIHCIHCIHCIHCIHCIHCCD8＋ cells were counted CD8＋ cells were counted using ImageJ softwareusing ImageJ softwareAverage of 3 hot spots Average of 3 hot spots were used for analysiswere used for analysisCD8＋ cell count in a hot spot : 181/ mm2 CD8＋ cell count in a hot spot : 181/ mm2 ImageJImageJCD8＋ cell count in a hot spot : 197/ mm2 CD8＋ cell count in a hot spot : 197/ mm2 ImageJImageJTumorTumorMucosaMucosaFFPE Tissue Kit（QIAGEN、Hilden、Germany）を使用して、プロトコルに従いDNAを抽出した。EBV関連腫瘍の診断 Epstein-Barr virus（EBV）のDNAの検出には、artus EBV PCR Kit（QIAGEN）を使用した。キットのプロトコルに従い、FFPE腫瘍部由来のDNAを鋳型としてStepOnePlus Real Time PCR System図2. 連続切片を用いた実験方法a） ホルマリン包埋 （FFPE）組織からの連続切片の作製b） 腫瘍内および腫瘍辺縁部におけるCD8＋リンパ球数のカウント方法の概要（Thermo Fisher Scientific、Waltham、MA）により、EBV-DNAの増幅の有無を確認した。わずかでも増幅が確認された症例は、さらにその切片からin situ hybridization（ISH）法によるEBV encoded RNA（EBER）の検出を行った。この検出にはBond Ready-to-Use ISH EBER Probe およびLeica BOND-Ⅲsystem（Leica Biosystems、Wetzlar、Germany）を使用し、マニュアルに従って4343
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