Decline of PD-L1 Immunoreactivity with Storage Duration in Formalin-Fixed Paraffin-Embedded Breast Cancer Specimens: Implications for Diagnostic Accuracy and Immunotherapy Eligibility in Triple-Negative Breast Cancer

Backgrounds: . Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is a critical predictive biomarker for immune checkpoint inhibitor (ICI) therapy in triple-negative breast cancer (TNBC). However, prolonged storage of formalin-fixed paraffin-embedded (FFPE) tissue may reduce antigenicity, potentially leading to false-negative results. We investigated the effect of FFPE storage duration on PD-L1 immunoreactivity. Methods. We retrospectively analyzed 63 TNBC cases with PD-L1 testing using the 22C3 pharmDx assay at diagnosis and repeated IHC on the same FFPE blocks after varying storage durations (<1, 1–2, 2–3, ≥3 years). PD-L1 positivity was defined as Combined Positive Score (CPS) ≥10. Associations with clinicopathologic features, pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC), and survival were evaluated. Results. At diagnosis, 41 patients (65.1%) were PD-L1–positive. In the PD-L1–positive group, decreased staining was observed in 0%, 11%, 13%, and 50% of cases for <1, 1–2, 2–3, and ≥3 years storage, respectively (p=0.015). No increased staining occurred. PD-L1 positivity correlated with higher Ki67 and nuclear grade but not with p53 status. pCR was achieved in 33% of PD-L1–positive vs 0% of PD-L1–negative NAC patients (p=0.0527). Survival analysis showed a non-significant trend toward shorter recurrence-free and overall survival in PD-L1–positive patients. Conclusions. Prolonged FFPE storage, particularly beyond three years, significantly reduces PD-L1 immunoreactivity. Testing on recent specimens is recommended to avoid false-negative results that may impact ICI eligibility.