Optimal Scoring of Brightfield Dual-Color In Situ Hybridization for Evaluation of HER2 Amplification in Breast Carcinoma: How Many Cells Are Enough?

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Objectives: The purpose of this study was to determine the optimum number of cells that should be counted when scoring human epidermal growth factor receptor 2 (HER2) brightfield dual-color in situ hybridization (BDISH), including cases with HER2/chromosome 17 (Chr17) ratios in the 1.80 to 2.20 range.

Methods: In total, 131 cases of breast carcinoma with HER2 immunohistochemistry and BDISH were included. For cases with a HER2/Chr17 ratio of less than 1.80 or more than 2.20 (n = 115), BDISH scoring was performed for 60 cells using three tumor fields, and for cases with a HER2/Chr17 ratio of 1.80 to 2.20 (n = 16), scoring was performed for 120 cells using six tumor fields. Mean HER2/Chr17 ratio and HER2 copy number were calculated for cumulative cell counts.

Results: The HER2 status as determined by the HER2/Chr17 ratio or HER2 copy number was unchanged following counting of additional cells in 100% of cases with ratio of less than 1.80 or more than 2.20. The HER2 status of two cases with ratios of 1.80 to 2.20 changed from positive to negative following counting of 120 cells.

Conclusions: Our findings support recommendations to score 20 nuclei in conjunction with careful assessment of immunohistochemistry and scan of the BDISH slide to identify areas of heterogeneity. Scoring of additional cells/fields is likely not of benefit and might be a disadvantage since the scorer moves out of the area of strongest signal.

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