Longitudinal evaluation of tumor-infiltrating lymphocyte scoring using automated region of interest registration in breast cancer.

backgroundTumor-infiltrating lymphocytes (TILs) are prognostic biomarkers in breast cancer (BC), particularly in HER2-positive and triple-negative subtypes. Assessment follows the international guidelines, in which pathologists evaluate whole hematoxylin and eosin (H&E)-stained slides while integrating representative regions of the invasive tumor. However, manual region selection can be labor-intensive, subjective, and may introduce variability, particularly across consecutive tissue sections. Automated region of interest (ROI) registration may mitigate this limitation, yet its impact on longitudinal TIL scoring has not been systematically evaluated. Here, we introduce three ROI registration strategies (direct, intermediate, and serial with/without quality control) and present an automated framework validated for consistent TIL scoring and clinical relevance in predicting relapse.

methodsWe analyzed 104 invasive BC cases, each with 12 consecutive H&E slides. A pathologist annotated ROIs on both the first and twelfth slides. We registered these ROIs using the proposed strategies. We then evaluated them with performance metrics, including Intersection over Union (IoU), Dice Similarity Coefficient (DSC), failure rate, and execution time. Two pathologists scored TILs on manual and automated ROIs. We assessed longitudinal consistency between the first manual ROI and the twelfth slide's corresponding ROIs. We also tested whether the association between TIL score and patient relapse outcomes was preserved.

resultsThe direct registration strategy achieved the highest geometric accuracy (mean IoU = 0.650, DSC = 0.769), with < 1% failure rate and ∼5.8 s execution time. TIL scores for both manual and automated ROIs closely matched (P = 0.84), showing strong agreement (Spearman's ρ = 0.923, ICC = 0.936, CCC = 0.915, Cohen's κ = 0.786). Longitudinal analyses showed no significant variability across distant sections (P = 0.79 and P = 0.62). TIL concentrations differed significantly between patients with and without relapse, and this association was preserved on both the first and twelfth slides (P < 0.05).

conclusionsWe present the first automated framework for longitudinal ROI registration to support TIL scoring in BC. By reducing manual effort and variability, the framework supports scalable evaluation of immune biomarkers across tissue depth while preserving clinically relevant signals, supporting precision immuno-oncology applications.