4.3. Validation via IHC Analysis

Prognostic implications of protein biomarkers, identified through proteomic analyses, were validated via IHC staining using a separate dataset consisting of chemotherapy-naïve, FFPE cancer tissues resected from the primary (non-metastatic) ovarian mass intraoperatively during debulking surgery (PDS cases) or diagnostic laparoscopy (NAC cases) (n = 107). After tissues were retrieved from the pathology archive of SNUH, they were histologically assessed through hematoxylin and eosin staining. To construct a TMA, three cores (2 mm in diameter) per patient were embedded in new recipient FFPE blocks using a trephine apparatus (Superbiochips Laboratories, Seoul, Korea).

IHC staining for AAT, NFKB, PMVK, VAP1, FABP4, PF4, APOA1, and AGP was performed using 4 μm thick TMA sections using a Benchmark autostainer (Ventana, Tucson, AZ, USA) in accordance with the manufacturer’s instructions (Table S8).

Because IHC staining of these eight antibodies and its prognostic effects was not previously evaluated in HGSOC, we determined the optimal cutoff values for each IHC staining, based on the sample distribution and prognostic significance (Table S8). Briefly, the extent (0–20%, 20–50%, 50–70%, 70–100%) and intensity (absent, weak, moderate, strong) of cytoplasmic/membranous immunoreactivity were semi-quantitatively assessed. Thereafter, the expression level of each protein was dichotomized into high versus low expression (Figure S4).