3D‐based approach for efficient analysis of immunostained histology images in evaluating downstream pathway activation in malignant tumors with molecular targets

In malignant tumors with molecular targets, the evaluation of downstream pathway activation, which could be heterogenous, may be important to predict and assess response to targeted therapy. Immunohistochemical staining is often used to examine the activation of multiple downstream markers in this setting while H&E staining provides the morphological and structural information to locate the tumor cells. With the evaluation of one immunostained slide at one time, it may be difficult to assess the simultaneous activation of multiple pathways within tumor cells, especially when reactive tumor cells are limited or scattered in the background of benign cells. By reconstructing the series of H&E and immuno stained images into a 3D composite image it would be easier to identify tumor cells that are reactive to multiple immunomarkers and assess the presence and/or heterogeneity of downstream pathway activation. In our experiment, serial sections of a lung adenocarcinoma with an EGFR mutation were obtained and stained with H&E and several immunomarkers, and then later scanned using the whole slide imaging in the laboratory. From the resulting 3D reconstructed image of the H&E and immunostained scanned images we could easily evaluate the expression of multiple immunomarkers within the tumor cells.