As an important therapeutic target in breast cancer, HER2 expression assessed by immunohistochemistry plays a critical role in breast cancer treatment. Recent advances in HER2 antibody–drug conjugate therapy have enabled patients with HER2-low expression breast cancer to benefit from the drugs. However, it is not known whether the HER2-low expression in breast cancer FFPE blocks would be lost as storage time increased. In this study, we aimed to assess the loss of HER2 antigenicity in stored FFPE blocks of breast cancer and the rescue effect of modifying the protocol of antigen staining. We selected archived HER2-low breast cancer FFPE blocks with stored time ranging from 1 year to over 15 years and re-detected the expression of HER2. Our study showed that HER2 antigenicity loss increased with storage time and could cause false negativity in HER2-low detection. Moreover, we showed that by either increasing the antigen retrieval time or applying the tyramide signal amplification (TSA) kit, the HER2 signal can be rescued and detected in about half of the cases with HER2-low loss without causing false positivity.

Aims Malignant effusion is a common manifestation of metastatic gastric cancer. This study compared the expression of key biomarkers (human epidermal growth factor receptor 2 (HER2), programmed cell death ligand 1 (PD-L1), claudin 18.2 (CLDN18.2) and fibroblast growth factor receptor 2b (FGFR2b)) between malignant effusion-derived cell blocks (CBs) and matched primary or metastatic tumour tissues and evaluated the longitudinal concordance of biomarker expression in serially collected CBs. Methods Biomarker status was retrospectively assessed by immunohistochemistry in CBs from malignant effusions and paired primary/metastatic gastric adenocarcinomas. HER2+ cases underwent fluorescence in situ hybridisation to confirm amplification. Results CLDN18.2 showed the highest positivity rate (34.0%). PD-L1 expression was positive in 8.6% of tumours and 13.9% of stroma. Lower rates were observed for FGFR2b (3.8%) and HER2 (3.3%). FGFR2b positivity was more frequent in CBs than in primary tumours (p=0.044) with higher expression levels (p=0.02). Conversely, CLDN18.2 positivity was lower in CBs versus primary tumours (p=0.004). Metastatic lesions showed higher CLDN18.2 positivity (p=0.04) and expression levels (p=0.002) compared with CBs. Serial CB analysis revealed high concordance rates: FGFR2b (99.1%), HER2 (98.1%), tumour PD-L1 (94.8%) and CLDN18.2 (92.2%). Conclusions Malignant effusion CBs show favourable intra-patient biomarker consistency over time, supporting their feasibility for longitudinal monitoring. Effusion-based testing shows potential value for potentially identifying candidates for FGFR2b-targeted therapies, pending further clinical validation.

Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive subtype with limited therapeutic targets. We retrospectively evaluated the expression of phosphatase and tensin homolog (PTEN) and transmembrane serine protease 4 (TMPRSS4) by immunohistochemistry in formalin-fixed, paraffin-embedded tumor tissues from 145 patients with histologically confirmed TNBC, and we analyzed associations with clinicopathological features, Fudan immunohistochemistry-based subtypes, and clinical outcomes. Clinicopathological analyses included all 145 cases. Exploratory survival analyses included 59 patients with complete, verifiable follow-up and outcome data (median follow-up, 46 months), during which three deaths and eight progression events occurred. PTEN-retained expression was associated with worse overall survival (OS; log-rank p = 0.030), whereas TMPRSS4-positive expression was associated with worse progression-free survival (PFS; log-rank p = 0.034). TMPRSS4 expression showed a nominal association with Fudan subtype in the unadjusted omnibus analysis (p = 0.025), which was considered exploratory after Benjamini–Hochberg correction (q = 0.379). The PTEN and TMPRSS4 staining categories were not significantly associated. In an exploratory four-group survival analysis, patients with concurrent PTEN-retained and TMPRSS4-positive expression (PTEN⁺/TMPRSS4⁺) showed the lowest OS and PFS estimates, patients with PTEN loss and TMPRSS4 negativity (PTEN⁻/TMPRSS4⁻) showed the highest estimates, and the two single-positive groups showed intermediate estimates. Independent transcript-level validation restricted to TNBC in The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) did not reproduce these protein-level associations. These protein-level findings should therefore be considered exploratory and hypothesis-generating and warrant confirmation in larger, adequately powered cohorts using standardized immunohistochemistry with complete treatment data.