Impact of stain variation and color normalization for prognostic predictions in pathology

In recent years, deep neural networks (DNNs) have demonstrated remarkable performance in pathology applications, potentially even outperforming expert pathologists due to their ability to learn subtle features from large datasets. One complication in preparing digital pathology datasets for DNN tasks is the variation in tinctorial qualities. A common way to address this is to perform stain normalization on the images. In this study, we show that a well-trained DNN model trained on one batch of histological slides failed to generalize to another batch prepared at a different time from the same tissue blocks, even when stain normalization methods were applied. This study used sample data from a previously reported DNN that was able to identify patients with early-stage non-small cell lung cancer (NSCLC) whose tumors did and did not metastasize, with high accuracy, based on training and then testing of digital images from H&E stained primary tumor tissue sections processed at the same time. In this study, we obtained a new series of histologic slides from the adjacent recuts of the same tissue blocks processed in the same lab but at a different time. We found that the DNN trained on either batch of slides/images was unable to generalize and failed to predict progression in the other batch of slides/images (AUC_cross-batch = 0.52 - 0.53 compared to AUC_same-batch = 0.74 - 0.81). The failure to generalize did not improve even when the tinctorial difference corrections were made through either traditional color-tuning or stain normalization with the help of a Cycle Generative Adversarial Network (CycleGAN) process. This highlights the need to develop an entirely new way to process and collect consistent microscopy images from histologic slides that can be used to both train and allow for the general application of predictive DNN algorithms.