CHAPTER 5 128 EnVision+Mouse HRP labelled polymer secondary antibody with DAB+ (K4006) was used. Slides were counterstained with Dako Automation Haematoxylin. Scoring of immunohistochemistry For ER and PR, the number of stained tumor nuclei regardless of staining intensity was scored, and subsequently cases were dichotomized, using 10% as a cut-off value. For L1CAM, the number of tumor cells showing membranous expression was scored and cases were dichotomized, using 10% as a cut-off value. For p53, staining was considered abnormal when there was complete absence of nuclear staining (null-expression) or when more than 80% of tumor cell nuclei showed strong expression (overexpression). For the Bergen subgroup, scoring was performed at the Haukeland university hospital and differed slightly from the Radboud staining protocols. In short, both intensity and area of positive tumour cells were scored. The intensity was scored from 0 (no staining) to 3 (strong), and the area as 0, 1 (<10%), 2 (10–50%) and 3 (51–100%). From this, a staining index (0–9) was calculated as the product of intensity and area. If heterogeneity was seen for the three tissue cylinders of each case, the three cylinders were given one overall averaged score. For L1CAM staining, index was dichotomized using ≥ 4 as a cut-off for positive expression. For ER staining index ⩽3 and PR staining index 0 was defined as negative expression. Pathologic expression of p53 (high) was defined as staining index ⩾4.
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