Diagnostic Value of Claudin-4 and EZH2 Immunohistochemistry in Effusion Cytology

Our plain-English summary of Diagnostic Value of Claudin-4 and EZH2 Immunohistochemistry in Effusion Cytology, by A. Elhosainy, M.M.A. Hafez, E.H. Yassin, et al (Asian Pacific Journal of Cancer Prevention, 2022).

The study

• Authors: A. Elhosainy, M.M.A. Hafez, E.H. Yassin, M. Adam, M.S. Elnaggar, N.A. Aboulhagag
• Journal: Asian Pacific Journal of Cancer Prevention
• Published: 2022;23(8):2779-2785
• Cited by: 7 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Metastatic adenocarcinoma (MAC) accounts for most cases of malignant effusions. Sometimes, it can be difficult to distinguish MAC from reactive mesothelial cells (RMC) in cytologic specimens. Our aim was to assess the diagnostic performance of a novel immunohistochemical panel composed of claudin-4 and EZH2 in differentiating MAC from RMC in effusion cytology.

Methods: A total of 80 cases of serous effusions (48 MAC and 32 RMC) were included. Immunohistochemistry using claudin-4 and EZH2 was performed on cell block sections of these cases. Assessment of staining patterns, intensity and percentage of target cells stained was done.

Results: Claudin-4 showed membranous staining in 46/48 of MAC and 1/32 of RMC. High EZH2 (≥ 50% of target cells) was detected in 42/48 MAC and 2/32 RMC. For the discrimination between MAC and RMC, claudin-4 exhibited 95.8% sensitivity and 96.9% specificity, high-EZH2 exhibited 87.5% sensitivity and 93.8% specificity, while the combination of both claudin-4 and high EZH2 showed 100% sensitivity and 90.6% specificity.

Conclusion: Claudin-4 shows high sensitivity and specificity in differentiation between MAC and RMC in effusion cytology, and might be useful as a solitary marker for MAC. Adding EZH2 to claudin-4 increases the sensitivity to 100%. However, the interpretation of EZH2 results can be challenging due to its focal expression in RMC and inflammatory cells.

In plain English

This retrospective laboratory study at Assiut University Hospital in Egypt tested whether two stains, claudin-4 and EZH2, help pathologists tell metastatic adenocarcinoma (cancer spread from elsewhere) apart from benign reactive mesothelial cells in fluid collected around the lungs, abdomen or heart. The authors stained cell samples from 80 effusions diagnosed between November 2019 and October 2021: 48 metastatic adenocarcinomas and 32 benign effusions. Mesothelioma was not one of the groups compared, and asbestos exposure was not studied. Claudin-4 was positive in 46 of 48 adenocarcinomas and 1 of 32 benign effusions (95.8% sensitivity, 96.9% specificity). High EZH2 staining, in at least 50% of target cells, was found in 42 of 48 adenocarcinomas and 2 of 32 benign effusions. The two markers together gave 100% sensitivity and 90.6% specificity. The authors concluded that claudin-4 might be useful as a single marker for metastatic adenocarcinoma in selected cases. They also concluded that adding EZH2 would raise sensitivity. However, they found EZH2 hard to interpret because it also stained some reactive mesothelial and inflammatory cells, and they did not advise using it alone.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Retrospective diagnostic study of immunohistochemical staining on cell block sections of effusion samples
• Who was studied: 80 serous effusion cases (45 pleural, 32 peritoneal, 3 pericardial) diagnosed between November 2019 and October 2021 at the Pathology Department of Assiut University Hospital, Egypt. There were 48 metastatic adenocarcinomas and 32 benign effusions with reactive mesothelial cells. Patients were 36 males and 44 females.
• Exposure: No asbestos or occupational exposure was studied. The tests evaluated were claudin-4 and EZH2 immunohistochemistry on cell block sections.
• Main results:
  • Claudin-4 was positive in 46 of 48 metastatic adenocarcinoma cases (95.8%).
  • Claudin-4 was positive in 1 of 32 benign effusions with reactive mesothelial cells.
  • For telling metastatic adenocarcinoma from reactive mesothelial cells, claudin-4 had 95.8% sensitivity, 96.9% specificity, 97.9% positive predictive value and 93.9% negative predictive value.
  • At a 5% cut-off, EZH2 was positive in 45 of 48 metastatic adenocarcinomas and 10 of 32 benign effusions. This gave 93.8% sensitivity, 68.8% specificity, 81.8% positive predictive value and 88% negative predictive value.
  • High EZH2, meaning staining in at least 50% of target cells, was found in 42 of 48 (87.5%) metastatic adenocarcinomas and 2 of 32 (6.3%) benign effusions. This gave 93.8% specificity and 87.5% sensitivity.
  • Positive claudin-4 combined with high EZH2 gave 100% sensitivity, 90.6% specificity, 94.1% positive predictive value and 100% negative predictive value.
  • EZH2 showed focal staining in inflammatory cells.
• The authors' conclusion: The authors concluded that claudin-4 shows high sensitivity and specificity for telling metastatic adenocarcinoma from reactive mesothelial cells in effusion cytology, and might be useful as a single marker in selected cases. They concluded that adding EZH2 would increase sensitivity, but that EZH2 results can be hard to interpret because of focal staining in reactive mesothelial and inflammatory cells. They did not advise using EZH2 alone.
• Limitations the authors state:
  • The authors found it problematic to choose a cut-off value for EZH2 positivity, because published values differ widely. They therefore tested both 5% and 50%.
  • The authors noted that EZH2 staining of inflammatory cells might cause diagnostic problems, especially when scattered malignant cells sit among many inflammatory cells.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Claudin-4, Immunohistochemistry, Cytology, Adenocarcinoma, Reactive Mesothelial Hyperplasia, Cell Block.

Related studies

• Claudin-4 immunohistochemistry is highly effective in distinguishing adenocarcinoma from malignant mesothelioma in effusion cytology — V.Y. Jo, E.S. Cibas, et al. (2014)
• Role of HEG1 and Claudin-4 Immunohistochemistry in the Differential Diagnosis of Lung Adenocarcinoma from Malignant Mesothelioma in Pleural Effusion Cytology — A.E. Abdelrahman, F.A. Elbadry, et al. (2025)
• Immunocytochemical utility of claudin-4 versus those of Ber-EP4 and MOC-31 in effusion cytology — T. Oda, S. Ogata, et al. (2016)
• The diagnostic utility of D2-40, calretinin, CK5/6, desmin and MOC-31 in the differentiation of mesothelioma from adenocarcinoma in pleural effusion cytology — T.S. Hyun, M. Barnes, et al. (2012)
• The Diagnostic Value of Immunohistochemistry and Biomarkers in the Diagnosis of Mesothelioma in Pleural/Peritoneal Effusions — S.M. Selvaggi (2026)
• EZH2, a unique marker of malignancy in effusion cytology — H. Jiang, R. Gupta, et al. (2014)

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