An Open-Source, Automated Tumor-Infiltrating Lymphocyte Algorithm for Prognosis in Triple-Negative Breast Cancer

Purpose: Although tumor-infiltrating lymphocytes (TIL) assessment has been acknowledged to have both prognostic and predictive importance in triple-negative breast cancer (TNBC), it is subject to inter and intraobserver variability that has prevented widespread adoption. Here we constructed a machine-learning based breast cancer TIL scoring approach and validated its prognostic potential in multiple TNBC cohorts. Experimental Design: Using the QuPath open-source software, we built a neural-network classifier for tumor cells, lymphocytes, fibroblasts, and "other" cells on hematoxylineosin (H&E)-stained sections. We analyzed the classifierderived TIL measurements with five unique constructed TIL variables. A retrospective collection of 171 TNBC cases was used as the discovery set to identify the optimal association of machine-read TIL variables with patient outcome. For validation, we evaluated a retrospective collection of 749 TNBC patients comprised of four independent validation subsets. Results: We found that all five machine TIL variables had significant prognostic association with outcomes (P <= 0.01 for all comparisons) but showed cell-specific variation in validation sets. Cox regression analysis demonstrated that all five TIL variables were independently associated with improved overall survival after adjusting for clinicopathologic factors including stage, age, and histologic grade (P <= 0.0003 for all analyses). Conclusions: Neural net-driven cell classifier-definedTIL variables were robust and independent prognostic factors in several independent validation cohorts of TNBC patients. These objective, opensource TIL variables are freely available to download and can now be considered for testing in a prospective setting to assess clinical utility.