Interobserver Agreement in Immunohistochemical Evaluation of Folate Receptor Alpha (FRα) in Ovarian Cancer: A Multicentre Study

Abstract

Folate receptor alpha (FRα) is a high-affinity folate transporter overexpressed in various epithelial malignancies, particularly high-grade serous ovarian carcinoma. Given its restricted expression in normal tissues and accessibility in tumors, FRα is an emerging therapeutic target. Immunohistochemistry (IHC) is the standard method for FRα assessment; however, interpretation is semi-quantitative and prone to interobserver variability. This study aimed to evaluate interobserver agreement among 12 pathologists in the IHC assessment of FRα in ovarian cancer, focusing on internal control adequacy, staining intensity, and the percentage of FRα-positive tumor cells. Thirty-seven high-grade serous ovarian carcinoma cases were stained using the VENTANA FOLR1 (FOLR1-2.1) RxDx Assay. A reference panel of four expert pathologists established consensus diagnoses. Twelve pathologists independently assessed the slides, recording internal control adequacy, staining intensity (positive vs. negative), and percentage of FRα-positive tumor cells. Interobserver agreement was measured using Fleiss' kappa and intraclass correlation coefficient (ICC). Agreement on internal control adequacy was almost perfect (κ = 0.84). Substantial agreement was observed for staining intensity (κ = 0.76), while percentage estimation showed almost perfect concordance (ICC = 0.89). Discrepancies were primarily confined to borderline cases (65-85% positivity) and tumors with intermediate staining, reflecting interpretive challenges near clinical decision thresholds. Pathologists demonstrated high reproducibility in FRα IHC assessment, particularly in estimating percentage positivity and control adequacy. These findings support the clinical utility of FRα IHC but underscore the need for standardized scoring criteria and potential integration of digital tools to enhance consistency, especially in borderline cases.

Keywords: VENTANA FOLR1; folate receptor alpha; high-grade serous ovarian carcinoma; immunohistochemistry; ovarian cancer.

Figure 1

Immunohistochemical determination of folate receptor 1 status. (A) Ovarian High-Grade Serous Carcinoma showing strong (3+) FOLR1 membrane positivity in >75% of tumor cells (H&E, 20×). (B) Ovarian High-Grade Serous Carcinoma showing circumferential and dot-like membranous staining of strong intensity (3+) in >75% of tumor cells (H&E, 20×). (C,D) Two examples of ovarian High-Grade Serous Carcinoma showing apical: the FOLR1 protein is localized to the apical (top) surface of neoplastic cells (H&E, 20×).

Figure 2

Immunohistochemical determination of folate receptor 1 status: examples of discordant cases. (A,B) Ovarian high-grade serous carcinoma showing FOLR1 membrane positivity (score 2+/3+) in 70% of tumor cells. This case has been considered borderline (65–85% of cells with a score of 2+ or 3+ positivity) ((A) H&E, 20×; (B) FOLR1-2.1 antibody on Benckmark Ultra platform, LSAB-HRP, 10×). (C,D) Another example falling within the borderline category: ovarian high-grade serous carcinoma showing FOLR1 membrane positivity (score 2+/3+) in 70% of tumor cells ((C) H&E, 10×; (D) Ventana FOLR1 RxDx Assay—FOLR1-2.1 antibody on (E,F) peritoneal metastasis of ovarian high-grade serous carcinoma showing FOLR1 membrane positivity not adequately evaluable, due to the diffuse crushing or heat-generated (cautery) artifacts ((E) H&E, 4×; (F) Ventana FOLR1 RxDx Assay—FOLR1-2.1 antibody on Benckmark Ultra platform, LSAB-HRP, 20×).

Figure 3

Immunohistochemical determination of folate receptor 1 status: examples of discordant cases. Distribution of estimated FRα-positive tumor cell percentages across borderline and discordant cases. Borderline cases were defined as tumors with 65–85% of neoplastic cells exhibiting 2+/3+ membranous staining, while discordant cases included tumors within the 31–45% and 46–64% percentage ranges that showed significant interobserver variability. Each boxplot represents the range, median, and variability of observer estimates for a given case.

Figure 4

Heatmap of individual observer estimates of FRα-positive tumor cell percentages across borderline and discordant cases. Borderline cases were defined based on a 65–85% staining threshold with 2+/3+ intensity, and discordant cases were selected among tumors showing marked variability in the 31–45% and 46–64% percentage intervals. Values highlight interobserver variability patterns.