(M0930-01-04) Cell Line–Anchored H-Score Strategy Defining Claudin-6 Expression Patterns in Ovarian Cancer

Purpose: Claudin-6 (CLDN6) is a key molecular marker involved in the diagnosis and treatment of various cancers, including ovarian cancer (1). Its limited expression in normal adult tissues and elevated levels in tumors make it an attractive target for therapeutic development (2). Immunohistochemistry (IHC) is the primary method for detecting Claudin-6 in formalin-fixed, paraffin-embedded (FFPE) tissues (3). However, the lack of standardized controls introduces variability in H-score interpretation, reducing the reliability of IHC results. This study aimed to develop a robust IHC scoring system for Claudin-6 using cell line–based controls and to establish an optimal cut-off value to enable consistent evaluation of Claudin-6 expression in clinical cancer samples.

Methods: The specificity and staining intensity of the Claudin-6 antibody were first validated through IHC staining of control cell lines (HEK293, A2780, OV-90, PA-01) exhibiting predefined negative, weak, moderate, and strong Claudin-6 expression. The Claudin-6 positivity cut-off value was defined as the H-score corresponding to the midpoint of the linear regression between cell line expression groups and H-scores. Subsequently, a comprehensive H-score analysis was conducted on tissue samples, including 95 ovarian cancer tissues, 15 normal ovarian tissues. The ratio of positive to negative expression of Claudin-6 in all tumors and each pathological type in ovarian cancer was determined according to the cut-off value. 

Results: Cell line–based controls confirmed the specificity of the Claudin-6 antibody, exhibiting consistent and distinct staining patterns. Following cell segmentation, H-scores were calculated using the DAB optical density of individual cells. A cut-off value of 27.5 was established as the midpoint (x = 0.5) of the linear regression between cell line expression levels and H-scores. In clinical tissues, Claudin-6 expression was significantly higher in ovarian cancer tissues compared to normal ovarian tissues. Among ovarian cancer subtypes, serous carcinoma showed markedly elevated Claudin-6 expression relative to mucinous and endometrioid carcinomas. Nonetheless, cases with minimal or absent Claudin-6 expression were also observed across ovarian cancer samples.

Conclusion: This study successfully established a standardized and quantitative IHC scoring system for Claudin-6 by control cell lines with gradually expression levels. The cell line-referenced H-score system, anchored by a data-driven cut-off value of 27.5, provided a robust and reproducible framework for assessing Claudin-6 expression in clinical samples. Application of this method to ovarian cancer tissues confirmed the elevated expression of Claudin-6 in tumors compared to normal tissues, particularly in serous ovarian carcinoma. Importantly, the identification of tumor cases with minimal Claudin-6 expression highlights the heterogeneity within and across cancer subtypes, underscoring the need for accurate quantification to inform targeted therapeutic strategies. This approach enhances the reliability and comparability of Claudin-6 IHC assessment and may serve as a practical reference for biomarker-driven cancer diagnostics and treatment selection.

References: 1. Du H, Yang X, Fan J, Du X. Claudin 6: Therapeutic prospects for tumours, and mechanisms of expression and regulation. Mol Med Rep. 2021;24(3):677. doi:10.3892/mmr.2021.12316
2. Reinhard K, Rengstl B, Oehm P, et al. An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors. Science. 2020;367(6476):446-453. doi:10.1126/science.aay5967
3. Mackensen A, Haanen JBAG, Koenecke C, et al. CLDN6-specific CAR-T cells plus amplifying RNA vaccine in relapsed or refractory solid tumors: the phase 1 BNT211-01 trial. Nat Med. 2023;29(11):2844-2853. doi:10.1038/s41591-023-02612-0

Acknowledgements: This study was funded by Accurant Biotech Inc. The authors declare no competing interests. All procedures involving human or animal materials were conducted in accordance with relevant ethical guidelines and approved protocols.


Representative Claudin-6 IHC staining in control cell lines and ovarian tissues.
Claudin-6 IHC was performed on FFPE sections of control cell lines (HEK293, A2780, OV-90, PA-01) and ovarian tissues. Staining intensity was scored as 0 (Negative) to +3 (Strong). Images show membrane-localized expression across different levels. Control cell lines served as staining and scoring references.


Establishing a Claudin-6 H-score cut-off using cell line–based reference standards.
QuPath-enabled cell segmentation of Claudin-6 IHC-stained slides allowed single-cell DAB intensity quantification. H-scores were calculated as: (1 × % 1+ cells) + (2 × % 2+ cells) + (3 × % 3+ cells). Linear regression of cell line expression groups (Negative to Strong) and corresponding H-scores (y = 88.00x − 16.50) defined a positivity cut-off of 27.5 at x = 0.5.


Claudin-6 H-score analysis and expression distribution in ovarian cancer.
(A) Claudin-6 H-scores in ovarian cancer tissues were significantly higher than in normal ovarian tissues (p < 0.05).
(B) Among histological subtypes, serous carcinoma showed the highest Claudin-6 expression.
(C) Distribution of Claudin-6-positive cases based on the 27.5 H-score cut-off. Overall, 20% of ovarian cancers were Claudin-6 positive, with serous carcinoma showing the highest positivity rate (33.3%).