Ancillary Studies in Mesothelioma Diagnosis: Distinguishing the Benign from the Malignant

Why Ancillary Studies Matter

Diagnosing mesothelioma can be incredibly challenging. The microscopic similarities between benign reactive mesothelial proliferations and malignant mesothelioma create a diagnostic minefield. That’s where ancillary studies step in—providing additional molecular and immunohistochemical markers that can differentiate benign from malignant mesothelial cells.

Several ancillary techniques have been explored, including:

• Immunohistochemistry (IHC)
• Proliferation markers
• Ploidy and flow cytometry
• Cytogenetics
• Electron microscopy (EM)

While some of these methods remain controversial, others—like IHC for epithelial membrane antigen (EMA)—have become cornerstones of diagnosis. Let’s dive deeper.

Epithelial Membrane Antigen (EMA): A Key Player

EMA is a membrane glycoprotein found in many normal epithelial tissues. But what makes it important in mesothelioma diagnosis?

• Malignant mesothelial cells tend to express EMA strongly, whereas benign mesothelial cells do not.
• Effusion cytology—a key method for detecting mesothelioma—relies on strong, thick, linear EMA membrane staining to confirm malignancy.
• However, weak EMA positivity in reactive effusions can lead to false positives.

How EMA Helps in Diagnosis

• Strong, thick, linear EMA membrane staining → Indicates malignant mesothelioma
• Weak or absent EMA staining → Likely reactive mesothelial proliferation (benign process)

Limitations of EMA

• Some benign effusions may show weak EMA positivity.
• EMA alone isn’t enough—it should be used alongside other markers.

Other Immunohistochemical Markers in Mesothelioma

While EMA is useful, it’s not infallible. Several other markers can help confirm malignant mesothelioma.

Key Immunohistochemical Markers

• BAP1 and MTAP loss are considered highly specific for malignant mesothelioma.
• Calretinin and CK5/6 are not specific, as they stain both benign and malignant mesothelial cells.

p53: A Potential Game-Changer

p53, a tumor suppressor gene, is frequently mutated in cancers. But does it help distinguish benign from malignant mesothelial cells?

• Esposito et al. (1995) found p53 positivity in 85.7% of mesotheliomas and 0% of reactive mesothelial proliferations.
• However, other studies have questioned its accuracy.

Should p53 Be Used?

• If p53 is strongly positive, malignant mesothelioma is likely.
• If p53 is negative, it doesn’t rule out malignancy.
• Some benign fluids have tested positive for p53, raising concerns about specificity.

So, while p53 immunostaining may be helpful, it’s not a standalone test.

Proliferation Markers: Can They Help?

Proliferating cell nuclear antigen (PCNA) is another potential marker.

• Higher PCNA levels have been found in malignant mesothelioma compared to benign mesothelial proliferations.
• However, overlap exists, making it less reliable than EMA or p53.

PCNA: A Useful Tool?

• Higher PCNA expression correlates with shorter survival.
• PCNA alone isn’t enough—it should be combined with other markers.

AgNORs: A Specialized Approach

Silver-positive nucleolar organizing regions (AgNORs) have been studied to differentiate benign vs. malignant mesothelial cells.

• Higher AgNOR counts are associated with mesothelioma.
• However, significant overlap exists between benign and malignant cases.

Bottom Line: AgNOR staining isn’t practical for routine diagnostics but may provide additional support in difficult cases.

Ploidy and Flow Cytometry: The Role of DNA Analysis

A cell’s ploidy refers to its DNA content. Most normal human cells are diploid (having two sets of chromosomes). However, malignant tumors often show aneuploidy (abnormal DNA content).

• 39–78% of mesotheliomas are diploid with a low S-phase fraction.
• Ploidy alone isn’t a reliable diagnostic marker, as some mesotheliomas appear diploid, and some benign proliferations can be aneuploid.

Does Flow Cytometry Help?

• Flow cytometry can analyze thousands of cells rapidly.
• However, its role in mesothelioma remains limited.

While ploidy studies can complement other tests, they aren’t definitive on their own.

Cytogenetics: A Look at Chromosomal Changes

Unlike many other cancers, mesothelioma doesn’t have a single characteristic genetic mutation. However, certain chromosomal deletions are common.

Key Chromosomal Abnormalities in Mesothelioma

• Deletions at 1p, 3p, 6q, 9p, and 22q are frequently observed.
• Loss of tumor suppressor genes plays a major role in mesothelioma development.

Future of Cytogenetics in Mesothelioma Diagnosis

• While cytogenetics isn’t used for routine diagnosis, new genomic sequencing techniques may revolutionize early detection.

Recommended Diagnostic Approach

When faced with a difficult case, how should pathologists proceed?

Stepwise Diagnostic Strategy

1. Start with effusion cytology.

   • If positive → Proceed to immunohistochemistry (EMA, BAP1, WT1).
   • If negative → Consider repeat sampling.

2. Perform a biopsy if necessary.

   • Thin core biopsy is minimally invasive.
   • Pleuroscopy with directed biopsy offers higher diagnostic yield.

3. Use multiple ancillary markers.

   • EMA, BAP1, and p53 provide high diagnostic accuracy.
   • Ploidy, AgNOR, and PCNA may offer additional insights.

4. Clinical correlation is crucial.

   • A patient’s asbestos exposure history can raise suspicion.
   • Follow-up imaging can track disease progression.

Key Takeaways

• EMA is a reliable marker, but not foolproof.
• p53 and BAP1 loss help confirm mesothelioma.
• PCNA, AgNOR, and ploidy studies have limited standalone value.
• Cytogenetics reveals important chromosomal deletions.
• A stepwise diagnostic approach improves accuracy.

Understanding ancillary studies is critical in mesothelioma diagnosis. By combining multiple techniques, pathologists can increase confidence in distinguishing benign mesothelial proliferations from malignant mesothelioma—ensuring patients receive the right diagnosis and appropriate treatment.