Diffuse mesothelioma (previously malignant mesothelioma) is a malignant neoplasm associated with asbestos exposure most commonly occurring in males. It arises predominantly in the pleura, though it can also occur in the peritoneum and pericardium, and it has a median survival of ~8–19 months for pleural cases, and even with therapy, the median survival is only ~6 months.

Clinically, it presents as recurrent pleural effusions (~90% of cases) with slowly increasing non-pleuritic pain, dyspnea and cough, and pneumothorax or hemothorax may occur. CXR shows pleural effusions and CT with contrast usually shows diffuse pleural thickening, with the tumor tending to form a rind encasing the lung. Treatment typically involves a multimodal approach, including surgical resection, chemotherapy, and radiation therapy, aimed at managing symptoms and controlling residual tumor. Less than 30% of patients are treated surgically.

Pleural fluid is usually bloody or serosanguinous, and very thick in consistency due to hyaluronan secreted by the cells. Cytologically is highly cellular, with a monotonous 3D population of mesothelial cells with variation in size that often can be distinguished from metastatic carcinomas by the absence of a second population of cells. There are 3 subtypes—epithelioid, sarcomatoid and biphasic, with epithelioid being notably the most common and the one that commonly sheds in effusions. Accurate diagnosis relies on a stepwise combination of cytologic, IHC, and molecular findings, which are critical for guiding treatment and prognosis. When a mesothelioma-related effusion contains adequate cellularity, an accurate diagnosis can be established in the majority of cases.

Epithelioid diffuse mesothelioma is characterized by tumor cells that are single or arranged in sheets, cohesive clusters, morules, papillary or tubule formations, generally with scalloped borders and can have a collagenous core. The tumor cells typically have a wide variation in size, with round, polygonal or cuboidal shapes and distinct cell borders. The cytoplasm is abundant, eosinophilic, dense, with possible inner lighter and outer darker staining, often vacuolated with perinuclear lipid vacuoles that stain for Oil Red O. They have low N:C ratio, can be multinucleated, with vesicular nuclei, finely granular chromatin, and prominent nucleoli. Mitoses are infrequent, and there is generally a lack of marked nuclear atypia or necrosis. Core biopsies frequently show well-differentiated epithelioid patterns, such as tubulo-papillary and glandular structures, separated by fibrous stroma. Recent studies advocate for a two-tier classification of epithelioid mesothelioma to better predict patient outcomes, categorizing cases into low or high grade based on nuclear features (including nuclear atypia and mitoses) and the presence of necrosis.

When the proliferation is morphologically consistent with mesothelioma, IHC is essential to confirm mesothelial origin, as primary pleural malignancies are rare, accounting for less than 10% of pleural tumors, with metastases being far more common. Tumor cells typically demonstrate positivity for mesothelial markers such as calretinin, WT1, CK5/6, D2-40, and HEG1, and can show positivity for EMA, while showing negativity for carcinoma markers like Claudin-4, MOC31, Ber-Ep4, CK7, CK20. They are also negative for other cells of origin, like TTF-1 and Napsin A, which are usually positive in lung adenocarcinomas. GATA3 is recognized to be positive in some mesothelioma cases, with references showing 23-58% of positivity.

Ancillary studies to identify specific mutations in mesothelioma can be performed after determining the cell of origin, as these mutations are highly indicative of malignancy and can help rule out reactive mesothelial cells, which are sometimes the major differential diagnosis. Common alterations include mutations in BAP1, CDKN2A, and NF2.

BAP1 mutations (somatic inactivations) are the most common encountered mutations in diffuse mesotheliomas and occur more frequently in the epithelioid type (70-80%). In patients with germline BAP1 mutations, exposure to even small amounts of asbestos increases risk of mesothelioma. BAP1 mutations can also be found in other tumors, such as clear cell renal carcinoma and cholangiocarcinoma. Homozygous deletions of CDKN2A (9p21) are found in 67-83% of epithelial and biphasic subtypes but are nearly universal (around 100%) in the sarcomatoid subtype, correlating with shorter survival rates; it is frequently co-deleted with the adjacent gene (9p21), which encodes the protein MTAP whose deficiency may increase sensitivity to PRMT inhibitors.

NF2 deletions and losses occur in 30-40% of cases but are not associated with specific subtypes or survival outcomes. TP53 mutations have been identified in ~8% of mesotheliomas and correlates with an aggressive clinical behavior and in younger populations. LATS2 mutations have also been found and are associated with poor prognosis.

In pleural and peritoneal mesothelioma, alterations can occur together and are not always linked to their genetic loci. BAP1 and CDKN2A can either co-occur or appear independently; however, CDKN2A loss appears to be less frequent in peritoneal mesotheliomas. Notably, TP53 alterations have been most frequently found in cases without BAP1 or CDKN2A alterations.

To evaluate these mutations and differentiate mesothelioma from benign proliferations:

1. BAP1: Loss of nuclear expression can be assessed through immunohistochemistry.
2. CDKN2A: The homozygous deletion at 9p21.3 can be identified by the cytoplasmic loss of MTAP through IHC, as a surrogate for the adjacent gene that is commonly lost. It can also be assessed by Fluorescence in situ hybridization (FISH), where a CDKN2A/CEP9 probe detects losses of the CDKN2A gene region or centromere of chromosome 9. Even though CDKN2A encodes for the tumor suppressor protein p16, it is important to note that IHC loss of p16 staining is not specific for CDKN2A homozygous deletion and is not recommended for this assessment. This is because p16 protein expression can be maintained despite p16 gene deletion and vice versa. MTAP IHC is a reliable surrogate to FISH and has shown complete correlation.
3. NF2: The protein encoded by NF2, Merlin, has been assessed as a surrogate marker of this mutation and thought to increase the diagnostic sensitivity. Complete loss of membranous expression can be evaluated through IHC, and is yet to be further validated. Some studies have detected NF2 fusions by FISH.
4. TP53: diffuse (>80%) p53 IHC is 100% specific for TP53 mutation and for malignancy, but a “null-mutant” p53 has not yet been identified in mesothelial lesions. Mutations in TP53 are not as commonly encountered but may be relevant in those with retained BAP1 and MTAP.

Loss of expression in these markers indicates a mutation; however, a positive internal control is necessary, as fixation can give a false negative, and assessment can be done only when cytology specimens have been validated, considering the antibody being used. While BAP1, MTAP IHC, and CDKN2A FISH are specific to distinguish mesothelioma from benign mesothelial proliferations (100%), they lack sensitivity (BAP1 and MTAP IHC sensitivity ranging from 24-65%). Next-generation sequencing can also be utilized to evaluate all relevant mutations and usually has high concordance with IHC, although a combination of IHC (BAP1, MTP, and recently with Merlin) has been shown a comparable sensitivity to NGS. Also, IHC might be better in some low-cellularity samples that may fall under the threshold for NGS detection and mutations that evade detection by NGS (loss of function rearrangements, deep-intronic splicing alterations). Alternatively, some mutations may spare the IHC epitope. NGS might be a useful tool in a high suspicion lesion with no detected alterations in IHC or FISH.