Protocol for Biomarker Ratio Imaging Microscopy with Specific Application to Ductal Carcinoma In situ of the Breast

This protocol describes the methods and steps involved in performing biomarker ratio imaging microscopy (BRIM) using formalin fixed paraffin-embedded (FFPE) samples of human breast tissue. The technique is based on the acquisition of two fluorescence images of the same microscopic field using two biomarkers and immunohistochemical tools. The biomarkers are selected such that one biomarker correlates with breast cancer aggressiveness while the second biomarker anti-correlates with aggressiveness. When the former image is divided by the latter image, a computed ratio image is formed that reflects the aggressiveness of tumor cells while increasing contrast and eliminating path-length and other artifacts from the image. For example, the aggressiveness of epithelial cells may be assessed by computing ratio images of N-cadherin and E-cadherin images or CD44 and CD24 images, which specifically reflect the mesenchymal or stem cell nature of the constituent cells, respectively. This methodology is illustrated for tissue samples of ductal carcinoma in situ (DCIS) and invasive breast cancer. This tool should be useful in tissue studies of experimental cancer as well as the management of cancer patients.