168 Chapter 13 Materials and methods A retrospective, proof of principle study was performed on a previously described cohort of patients with adenocarcinoma of the lung (diagnosed on biopsies and 195 resections from different institutes submitted originally for mutation analysis), between November 2007 and November 2010 at the VU University Medical Center181. Follow-up was retrieved from patient files. Hematoxylin and eosin (H&E) stained slides were retrieved from the archive. Cytokeratin 7 (CK7) The immunohistochemical stain for CK7 (clone OVTL12/30 (Agilent/Dako (Glostrup, Denmark, catno. M701801) was performed in a Roche/Ventana benchmark Ultra (Roche, Basel Switzerland) with 3µm tissue slides mounted on TOMO-glass slides (Roche, Basel, Switzerland). Antigen retrieval was applied with high pH-buffer CC1(32 minutes at 100°C) and the antibody was diluted 1/100 (incubated for 32 minutes at 36°C) and detected with the Optiview DAB kit under standard conditions. Sections were dehydrated with ethanol 100%, cleared with Xylene and coverslipped with Tissue-Tek coverslip film on the Sakura coverslipper (Sakura Finetek Europe B.V, Alphen aan den Rijn, The Netherlands). Internal positive controls were pneumocytes and respiratory epithelial cells. Internal negative controls were e.g., smooth muscle cells in vessel walls, bronchioli. Morphologic evaluation A first scoring of H&E-stained slides was performed based on pattern recognition as advocated according to the WHO by one pathologist (HB). Subsequently, a consensus score was made by two pathologists incorporating surgical collapse (HB, ET). Surgical collapse with AIS was characterized as compressed lung tissue where tumor cells were lining compressed alveolar walls with reduced or minimal air remaining. This pattern has a regular, occasionally streaming appearance with a monolayer of tumor cells. Tangential cutting of tumor cells (focal seemingly > 2 cell layers in an area otherwise fitting with collapsed AIS) was interpreted as collapse and not classified as ‘focally > 2 cell layers’. Surgical collapse was taken into account to interpret some seemingly papillary or acinar patterns as non-invasive. After reading of H&E and a washout period of >2 weeks the CK7 slides were scored for the same items as the H&E. The assumption was that most adenocarcinoma cases were CK7 positive and when lined on alveolar walls a consistent architectural pattern could be present and be supportive of non-invasive growth. In addition, characteristics of invasive growth such as individual tumor cells, and more luminal proliferation than fitting with AIS, such as prominent multilayering, cribriform and/or solid growth we decisive for invasion.
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