Supplementary Material for: Prognostic impact of immunophenotypic variation in subcapsular sinus endothelium of sentinel lymph nodes in invasive breast carcinoma

Introduction Several studies demonstrated the de novo-formation of lymphatic vessels in tumor-draining lymph nodes (LN), partly preceding lymphatic metastases. This “lymphovascular niche” supposedly facilitates survival of metastatic tumor cells. This study aims at evaluating the previously observed immunophenotypic variations of subcapsular endothelial cells (SEC) in a larger cohort by software-assisted image analysis. Methods Suitable cases with sentinel-LN (SLN) of invasive breast cancer were identified in the Institute of Pathology, corresponding data was extracted. LN of 231 patients were stained for HE, D2-40, CD31 and Prox1. QuPath software was used for assessing the immunohistochemical stained area of endothelial cells of the subcapsular sinus. The Cutoff Finder web application was used for identification of the best cutoff for continuous parameters according to overall survival (OS). Collected data was statistically evaluated for available data. Results A larger area of CD31-positive SEC was significantly associated with worse OS (p=0.001), as was a higher proportion of D2-40-stained subcapsular sinus (p=0.045). Larger area of D2-40-/CD31-/Prox1-positive SEC and higher proportion of D2-40 stained subcapsular sinus were independent marker for worse OS in multivariate analysis in the whole cohort, for D2-40- and CD31-positive SECs as well as higher proportion of D2-40-stained sinus including nodal-negative status, respectively. Conclusion QuPath-assisted evaluation of immunophenotypic variation in subcapsular sinus endothelium in SLN essentially confirmed and extended our previous findings. Larger positive area of D2-40 and CD31-positive SECs emerged as a strong independent negative prognostic factor, even before evident nodal metastasis. The potential function of alterations in D2-40-/CD31-expression in SECs has yet to be elucidated.