Slide Selection Introduces Sampling-Induced Prediction Uncertainty in Digital Pathology AI: Evidence from Multi-Slide Breast Cancer Cohorts

Background/Objectives: Digital pathology models increasingly infer molecular phenotypes from hematoxylin and eosin whole-slide images (WSIs), but most assume that one slide adequately represents patient-level tumor biology. We quantified prediction instability caused by slide selection and evaluated multi-WSI aggregation. Methods: Six multiple-instance learning architectures were developed in task-specific subsets of TCGA-BRCA (source cohort: 1006 patients and 1065 WSIs) and independently evaluated in 122 patients (400 WSIs) from CPTAC-BRCA. The clinically realistic comparison was random one-slide-per-patient inference versus patient-level aggregation of all available WSIs. Label-conditioned best- and worst-slide analyses were used only as retrospective oracle bounds. The recurrence-risk endpoint was a research-derived 21-gene recurrence-score surrogate calculated from RNA sequencing rather than a clinically reported Oncotype DX result. Results: Random single-slide selection yielded AUCs of 0.77–0.86 for recurrence-risk prediction and 0.65–0.77 for HER2 status. Patient-level multi-WSI aggregation yielded AUCs of 0.81–0.89 and 0.71–0.80, respectively. The architecture-controlled AUC improvement from random selection to aggregation ranged from 0.026 to 0.041 for recurrence risk and from 0.036 to 0.073 for HER2. Aggregation also reduced Brier scores by 0.010–0.028 and 0.009–0.106, respectively. The label-conditioned oracle analyses demonstrated wider theoretical performance ranges of 0.46–0.97 and 0.40–0.92, respectively. Slide-selection sensitivity persisted after excluding tumor-evidence-negative and low-tumor-content WSIs. Conclusions: Slide selection is a material source of sampling-dependent prediction uncertainty. Patient-level multi-WSI aggregation mitigated selection-dependent degradation across architectures, although validation using routine institutional material and clinically reported molecular assays remains necessary.