Fig. 2

Evaluation of RIS, Tumor budding and TLS according by hematoxylin/eosin (H&E) staining and immunohistochemistry (IHC). A Representative sections for evaluating RIS, Tumor budding and TLS. B–D Representative images of TLSs in each maturation category as evaluated by H&E. E–G Representative images of Tumor budding in each category as evaluated by H&E. H–I Representative immunohistochemistry of CD8 + cells stained with CD8 antibody and FOXP3 + cells stained with FOXP3 antibody. J The RIS scoring system is based on the counting of two lymphocyte subsets (CD8 and FOXP3) in two locations (CT and IM) of the primary tumor. All patients were divided into high (H in a dark circle) or low (L in a light circle) groups for the density of each marker in each region. A high density of immune cells was recorded as a score of 1, and a low density was recorded as a score of 0. Patients were stratified according to a score of 0, 1–3 or 4, representing low, intermediate, or high RIS, respectively. For example, score 0 refers to a tumor with low densities of CD8 + and FOXP3 + cells in CT and IM regions. Score 4 refers to a tumor with high densities of CD8 + and FOXP3 + cells in CT and IM regions. TLS, tertiary lymphoid structures; E-TLS, early-stage TLS; PFL-TLS, primary follicle-like stage TLS; SFL-TLS, secondary follicle-like stage TLS; CT, center of the tumor; IM, invasive margin; RIS, regulatory-Immunoscore