Digital image analysis–based quantification of p53 immunohistochemistry in gastric cancer: a systematic comparison of three-tier cutoff-derivation strategies in tissue microarrays

Background: p53 immunohistochemistry (IHC) is a common surrogate for TP53 alteration, but in gastric cancer, its concordance is imperfect and depends on a non-standardized cutoff. Digital pathology enables H-score quantification, yet neither its reproducibility nor the choice of cutoff- derivation strategy has been systematically compared. Methods: A semi-automated digital workflow quantified p53 IHC in tissue microarrays from 528 gastric cancer patients. Tumor cells were detected with a deep-learning model (InstanSeg), and nuclear staining was expressed as a continuous H-score (0-300). Reproducibility was assessed at each observer-dependent step. Five thresholding strategies were compared: manual cutoffs, tertiles, log-rank optimization, the extended Youden index, and volume under the receiver operating characteristic surface (VUS). The latter two strategies used the manual three-tier IHC interpretation as the reference standard. Results: Quantification was reproducible: two independent manual interpretations agreed in 91.7% of cores (κ = 0.845). The H-score was robust to the detection algorithm (intraclass correlation coefficient, 0.990) and to repeated threshold determination (0.954). H-scores were right-skewed (median, 45.5), and the strategies yielded distinct cutoffs (8.0-27.6 and 78.6-106.0). Log-rank optimization provided the widest separation but lost significance after multiplicity adjustment (p = .168). The extended Youden and VUS cutoffs were statistically indistinguishable. As a supporting check, all classifications showed nominal overall-survival separation, with low and overexpression faring similarly worse than wild-type. Conclusions: Digital p53 IHC quantification was reproducible at each observer-dependent step. The extended Youden and VUS criteria performed equivalently; therefore, we propose Youden as a reasonable default, pending validation and TP53 correlation.