Prevalence and Clinical Significance of Claudin-3 Expression in Cancer: A Tissue Microarray Study on 14,966 Tumor Samples

Academic Background

Claudin-3 (Cldn3) is a member of the Claudin family, involved in the formation of tight junctions (TJs), which regulate intercellular permeability. TJs play a crucial role in maintaining the barrier function of epithelial and endothelial cells. Although Claudin-3 is widely expressed in normal epithelial cells, its altered expression has been linked to tumor progression and prognosis in various cancers. Despite its widespread presence in normal cells, Claudin-3 is considered a potential drug target due to its increased accessibility in cancer cells, which exhibit less orchestrated growth.

However, studies on the expression and clinical significance of Claudin-3 in cancer have yielded inconsistent results. Variations in antibodies, immunohistochemistry (IHC) protocols, and criteria for positivity have led to significant discrepancies in reported Claudin-3 positivity rates. To better understand the prevalence and potential clinical significance of Claudin-3 in cancer, this study conducted a comprehensive IHC analysis using tissue microarray (TMA) technology on 14,966 tumor samples from 133 different tumor types and subtypes, as well as 76 normal tissue samples.

Source of the Paper

This study was conducted by Seyma Büyücek and colleagues from the Institute of Pathology at the University Medical Center Hamburg-Eppendorf, the Institute of Pathology at the Clinical Center Osnabrück, and the Department of Pathology at the Academic Hospital Fürth, Germany. The paper was published in 2024 in the journal Biomarker Research, titled Prevalence and clinical significance of claudin-3 expression in cancer: a tissue microarray study on 14,966 tumor samples.

Research Process

1. Tissue Microarray (TMA) Construction

The study utilized two TMAs: one containing 608 samples from 76 normal tissue types, and the other containing 14,966 primary tumor samples from 133 tumor types and subtypes. All samples were obtained from the archives of the Institute of Pathology at the University Medical Center Hamburg-Eppendorf, the Institute of Pathology at the Clinical Center Osnabrück, and the Department of Pathology at the Academic Hospital Fürth, Germany. Samples were fixed in 4% buffered formalin and embedded in paraffin. Each patient was represented by a single tissue spot with a diameter of 0.6 mm in the TMA.

2. Immunohistochemistry (IHC) Analysis

Freshly cut TMA sections were subjected to IHC staining in a single day. Sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in Tris-EDTA-citrate buffer (pH 7.8) at 121°C. Endogenous peroxidase activity was blocked, and staining was performed using a rabbit recombinant monoclonal antibody against Claudin-3 (HMV-309). Staining was visualized using the DAB detection system, and sections were counterstained with Hemalaun.

3. Data Analysis

IHC results were scored by a single pathologist, with staining intensity categorized as 0, 1+, 2+, or 3+. Statistical analyses were performed using JMP17® software. The chi-square test was used to assess associations between Claudin-3 expression and tumor phenotype, while Kaplan-Meier curves and the log-rank test were used to evaluate survival differences.

Key Findings

1. Claudin-3 Expression in Normal Tissues

Claudin-3 expression in normal tissues was predominantly membranous, with strong staining observed in luminal cells of breast glands, prostate, thyroid follicular cells, respiratory epithelial cells, salivary gland cells, small intestine and colorectal epithelial cells, bile duct epithelial cells, pancreatic acinar cells, renal collecting ducts, endometrial gland cells, and other tissues.

2. Claudin-3 Expression in Tumor Tissues

Of the 12,314 analyzable tumor samples, 68.9% exhibited Claudin-3 positivity, with 11.6% showing weak, 6.2% moderate, and 51.1% strong staining. Claudin-3 positivity was most frequently observed in neuroendocrine neoplasms (92-100%), adenocarcinomas (67-100%), tumors of the female genital tract (e.g., ovarian and endometrial carcinomas, up to 100%), and various subtypes of breast cancer (95.3-100%). In contrast, Claudin-3 expression was less common in squamous cell carcinomas (0-43.2%) and largely absent in melanoma, mesenchymal neoplasms, and hematolymphatic tumors.

3. Association Between Claudin-3 Expression and Tumor Prognosis

In clear cell renal cell carcinoma (ccRCC), low Claudin-3 expression was strongly associated with poor ISUP grade (p < 0.0001), Fuhrman grade (p < 0.0001), Thoenes grade (p < 0.0001), advanced pathological stage (p < 0.0001), high UICC stage (p = 0.0006), distant metastasis (p = 0.0011), and shortened overall (p = 0.0118) and recurrence-free survival (p < 0.0001). In papillary renal cell carcinoma (pRCC), low Claudin-3 expression was also associated with poor grade (p < 0.05), advanced pathological stage (p = 0.0273), and distant metastasis (p = 0.0357). In urothelial carcinoma, high Claudin-3 expression was linked to high grade (p < 0.0001) and nodal metastasis (p = 0.0111).

Conclusion

This study provides a comprehensive assessment of Claudin-3 expression across a wide range of tumor types using tissue microarray technology. The results demonstrate significant levels of Claudin-3 expression in many tumor entities and identify reduced Claudin-3 expression as a potential prognostic marker in renal cell carcinoma, particularly ccRCC.

Research Highlights

1. Large-Scale Sample Analysis: The study analyzed 14,966 tumor samples across 133 different tumor types and subtypes, offering a comprehensive dataset on Claudin-3 expression in cancer.
2. Claudin-3 as a Prognostic Marker: The findings suggest that Claudin-3 expression levels are strongly associated with prognosis in clear cell renal cell carcinoma, indicating its potential as a prognostic marker.
3. Tumor-Type-Specific Expression: Claudin-3 expression was highest in neuroendocrine neoplasms, adenocarcinomas, and tumors of the female genital tract, while being less common in squamous cell carcinomas and hematolymphatic tumors, highlighting its tumor-type-specific expression.

Research Significance

This study not only provides extensive data on Claudin-3 expression in cancer but also reveals its potential prognostic value in renal cell carcinoma. These findings offer a theoretical foundation for future development of cancer therapies targeting Claudin-3. Additionally, the study underscores the importance of standardized IHC protocols in cancer research to minimize discrepancies between studies.

Additional Valuable Information

The study validated the specificity of the IHC analysis by comparing results obtained with different antibodies and RNA expression data, further enhancing the reliability of the findings. In the future, Claudin-3 may emerge as a significant target for cancer diagnosis and treatment, particularly in specific tumor types such as renal cell carcinoma.