Guidelines for Pathologic Diagnosis of Malignant Mesothelioma: 2012 Update of the Consensus Statement from the International Mesothelioma Interest Group

The diagnosis of MM should always be based on the results obtained from an adequate biopsy (less commonly cytology, exfoliative and fine-needle aspiration) in the context of appropriate clinical, radiologic, and surgical findings. A history of asbestos exposure should not be taken into consideration by the pathologist when diagnosing MM. Location of the tumor (pleural versus peritoneal) as well as the sex of the patient will affect the differential diagnosis as discussed below. The histologic diagnosis of MM is based not only on the appropriate morphology but also on the appropriate immunohistochemistry. Specific information on antibody clones and their source should be obtained from the current literature, since this is an evolving area and is outside of the scope of this article. Molecular testing is now more widely available and is helpful in selected cases.

Separating benign from malignant mesothelial proliferations presupposes first that the process has been recognized as mesothelial. The diagnostic approach used when distinguishing reactive mesothelial hyperplasia from epithelioid mesothelioma is different from that used when distinguishing fibrous pleuritis from desmoplastic mesothelioma.²  The major problem areas are discussed below.

It is well known that reactive mesothelial proliferations may mimic mesothelioma (or metastatic carcinoma). Some of the causes of reactive mesothelial hyperplasia in the pleural space include infections, collagen vascular diseases, pulmonary infarcts, drug reactions, pneumothorax, subpleural lung carcinomas, surgery, trauma, and nonspecific inflammation. Exuberant mesothelial reactions also are encountered in the peritoneum and pericardium, and the latter may be particularly worrisome.

The specific features of a reactive mesothelial proliferation that may mimic a neoplasm include high cellularity, the presence of numerous mitotic figures and cytologic atypia, the presence of necrosis, the formation of papillary groups, and entrapment of mesothelial cells within fibrosis mimicking invasion (Figure 1). Features distinguishing reactive mesothelial hyperplasia from mesothelioma are summarized in Table 1.

Demonstration of stromal or fat invasion is a key feature in the diagnosis of MM (Figure 2). Invasion may be into visceral or parietal pleura (or beyond) and this can be highlighted with immunostains such as pancytokeratin or calretinin. Invasion into the peripheral lung is also a useful feature in cases involving visceral pleura. Invasion by mesothelioma is often subtle and may be into only a few layers of collagenous tissue below the mesothelial space. Invasive mesothelial cells may also be deceptively bland in appearance and completely lack a desmoplastic reaction. However, it is emphasized that when a substantial amount of solid malignant tumor with histologic features of MM (ie, a tumor mass) is identified, the presence of invasion is not required for diagnosis.

Reactive mesothelial proliferations tend to show a uniformity of growth and this may be highlighted with pancytokeratin staining, which shows regular sheets and sweeping fascicles of bland spindle cells that respect mesothelial boundaries in contrast to the disorganized growth and haphazardly intersecting proliferations seen in mesothelioma. The use of pancytokeratin staining to assess the overall architecture of a mesothelial proliferation cannot be overemphasized.

While certain immunohistochemical stains are more likely to show positivity in benign proliferations and others in malignant proliferations, they should not be solely relied on for diagnosis in individual cases (Table 2). The most helpful of these include epithelial membrane antigen (EMA), p53, desmin, glucose transporter 1 (GLUT-1), and insulin-like growth factor II messenger RNA–binding protein 3 (IMP3), which can be applied as a panel.^3–6 When GLUT-1 staining is positive, it may be a helpful marker for MM, both epithelial and sarcomatoid (Figure 3, A through D) but is not helpful when negative. It is more likely to be positive in pleural than in peritoneal MM. Overall sensitivity and specificity are reported to be 54% and 98%, respectively.⁷  Oncofetal protein IMP3 was recently shown to be positive in 33 of 45 MMs (73%; Figure 4) and negative in all 64 reactive mesothelial lesions tested.⁸ 

Markers that have been studied in the literature and that are not considered useful in the diagnosis of mesothelioma versus mesothelial hyperplasia include minichromosome maintenance 2 protein, telomerase transcriptase expression (which needs to be studied further), Ki-67, transforming growth factor, epidermal growth factor receptor, Bcl-2, and argyrophilic nucleolar organizer region.⁹ 

Most recent published studies show that the presence of homozygous deletion of p16 (as discussed below) rules out a reactive lesion.

The identification of features of malignancy in a desmoplastic mesothelioma requires adequate tissue, and the amount of tissue in a closed pleural biopsy is often insufficient. Large surgical biopsy specimens are generally needed. High-grade sarcomas presenting in the pleura generally do not enter into the differential diagnosis of fibrous pleurisy versus desmoplastic mesothelioma. Features to separate the latter two are shown in Table 3.

The distinction of fibrous pleurisy from desmoplastic mesothelioma can be made by identifying one or more of the following features in a spindle cell proliferation of the pleura: invasive growth, bland necrosis, frankly sarcomatoid areas, and metastatic disease.¹⁰  Stromal invasion is often more difficult to recognize in spindle cell proliferations of the pleura than in epithelioid proliferations. The invasive malignant cells are often deceptively bland, resembling fibroblasts, and pancytokeratin staining is invaluable in highlighting the presence of cytokeratin-positive malignant cells in regions where they should not normally be present: in the connective tissue, adipose tissue, or skeletal muscle deep to the parietal pleura, or invading the visceral pleura and lung tissue (or other extrapleural structures present in the sample) (Figure 5, A and B). Bland necrosis of paucicellular fibrous tissue may be subtle and one may be reluctant to base a diagnosis of malignancy solely on its presence. Fortunately, most cases that show bland necrosis also show invasive growth.¹⁰  Similarly, the presence of “frankly sarcomatoid foci” is a distinctly subjective determination and one would be reluctant to base a diagnosis of malignancy on its presence alone, since reactive processes may show marked cytologic atypia, albeit typically at the surface of the process.

While identification of invasion into adjacent tissues is often straightforward with the aid of pancytokeratin staining, Churg et al¹¹  have recently pointed out that fatlike spaces (“fake fat”) may be encountered in some cases of organizing pleuritis, probably as a result of artifactual changes in the dense fibrous connective tissue (Figure 6, A and B). In these regions, horizontally oriented cytokeratin-positive cells may be encountered around the fatlike spaces (Figure 7). Awareness of this phenomenon, and looking for vertically oriented cytokeratin-positive cells invading into readily identifiable adipose tissue (Figure 5, B), should help one avoid misinterpreting this phenomenon. Also, S100, laminin, and collagen IV usually show positivity in true adipose tissue and can help in distinguishing it from fake fat, which is negative for all three (Figure 8, A through F).

Uniformity of growth and thickness of the pleural process, surface atypia with deep maturation, and perpendicular thin-walled vessels are all typical of reactive fibrous pleuritis (Figure 9, A and B), in contrast to the disorganized growth pattern and variable thickness of desmoplastic mesotheliomas. A helpful clue in desmoplastic mesotheliomas is the presence of expansile nodules of varying sizes with abrupt changes in cellularity between nodules and their surrounding tissue.

Mesotheliomas often present with recurrent serous effusions, which are submitted for cytologic evaluation. Even though the cytologic features of MM were described more than 50 years ago and have been further refined in numerous subsequent articles, there is still doubt as to the ability of the cytopathologic modality to establish a definitive diagnosis of malignant mesothelioma.^12,13  The published sensitivity of cytologic diagnosis of mesothelioma ranges between 32% and 76%. This broad range of sensitivity (high false-negative rate) is probably related to sampling rather than interpretation, though one has to accept that there is a broad morphologic overlap between reactive mesothelial cells and malignant cells of mesothelioma. The absence of one of the key histologic diagnostic features of malignant mesothelioma, invasion of preexisting tissue (not granulation tissue), is not a characteristic of exfoliative cytology specimens.

To achieve correct cytologic diagnosis it is important to obtain an adequate amount of well-preserved fluid, which has to be prepared to ensure satisfactory cell concentration suitable for making quality smears (direct, cytospin, thin-layer) and cell blocks. Similar to histologic specimens (as discussed in other sections of this article), application of immunocytochemical and molecular techniques, either on smears or on cell blocks, enhances greatly the possibility to reach a correct diagnosis.^8,14–17  Molecular techniques, such as fluorescence in situ hybridization (FISH) in demonstrating homozygous deletion of the p16 gene in about 70% of mesothelial proliferations, are particularly promising, as reported specificity is 100%.^18–20  However, emerging data indicate that subtyping of epithelioid mesothelioma according to morphologic features and nuclear grade²¹  is important to predict survival, hence a cytologic diagnosis of “malignant mesothelioma epithelioid type” might not be sufficient in the future. Apart from diagnostic difficulties, the frequent practice of litigation in cases of mesotheliomas makes pathologists reluctant to diagnose mesothelioma without histologic confirmatory evidence.

One also has to recognize that not all mesotheliomas yield effusions and the sarcomatoid mesotheliomas are virtually never diagnosed on effusion cytology. In such cases, fine-needle aspiration, combined with core biopsy (or larger tissue samples), are necessary to establish the diagnosis.

Many of the cytologic features (scalloped borders of cell clumps, intercellular windows, with lighter dense cytoplasm edges, and low nuclear/cytoplasmic ratios) are shared between reactive and malignant epithelioid mesothelial cells. Usually, the malignant cells in sarcomatoid MM are not shed into the effusion fluid, which may contain the overlying reactive epithelioid mesothelial cells that may mislead the pathologist.

The most useful cytologic features of epithelioid MMs are as follows (Figure 10, A through D):

• The presence of numerous relatively large (>50 cells) balls of cells with berrylike external contours is characteristic of MM. Most cells are much larger than the average mesothelial cells. This includes enlargement of cytoplasm, nucleus, and nucleolus.²² 

• The presence of macronucleoli. However, prominent nucleoli can be present in reactive mesothelial cells and not all MM cells have macronucleoli.

• Nuclear atypia, if present.

Key cytologic features of adenocarcinoma are as follows:

• Clumps of cells usually have smooth rather than berrylike borders.

• The nuclear to cytoplasmic ratio is usually higher than in MM

• Nuclear variability in shape and size is much more common.

• Cytoplasmic vacuoles often contain epithelial mucin in contrast to mesothelial cells, which contain hyaluronic acid.

• Cytoplasm is less dense than in mesothelial cells, and “windows” are rarely present.

• •

  Psammoma bodies (when present) are more likely to be a feature of adenocarcinoma than MM, but they do occur in MM rarely.

The differential diagnosis, and use of immunohistochemistry and molecular markers in cytologic specimens, is similar to that in tissue sections (see below).

Most MMs are readily identified or strongly suspected on routine hematoxylin-eosin staining where they exhibit a variety of histologic subtypes, broadly divided into epithelioid, sarcomatoid, or mixed (biphasic) categories. Multiple patterns of each of these subtypes have been described, some of which are now being shown to have prognostic importance (see below). Also, the recognition of the various patterns is helpful for the pathologist diagnostically and will guide the differential diagnosis and selection of appropriate markers. However, most mesotheliomas have several patterns and on a biopsy sample it may not be possible to further subclassify the tumor. Thus the pattern may be included as a comment or in the microscopic description (Table 4). Although histologic grading has not traditionally been performed, a recent study of resected epithelioid MM²³  showed that a 3-tiered nuclear grading score based on mitotic activity and nuclear atypia is strongly predictive of survival. It will be interesting to see if these results are corroborated in future studies.

Epithelioid MMs are composed of polygonal, oval, or cuboidal cells that often mimic nonneoplastic reactive mesothelial cells. Sarcomatoid MMs usually consist of spindle cells but can be composed of lymphohistiocytoid cells and/or may also contain heterologous rhabdomyosarcomatous, osteosarcomatous, or chondrosarcomatous elements.^24,25  Mixed or biphasic MMs contain both epithelioid and sarcomatoid areas within the same tumor.^26–32  In general, the differential diagnosis for MM depends on its basic histologic category: the differential diagnosis for epithelioid MM includes carcinomas and epithelioid cancers; the differential diagnosis for sarcomatoid MM includes sarcomas and other spindle cell neoplasms; and the differential diagnosis of mixed MM includes mixed or biphasic tumors such as synovial sarcoma and metastatic pleomorphic carcinoma of lung. Desmoplastic mesotheliomas may mimic fibrous pleuritis. Since each broad histologic category has its own distinctive differential diagnosis, the immunostains selected for further workup of an MM are dictated by the histologic category into which it falls.³³ 

The most frequent histologic type of MM is epithelioid. The common secondary growth patterns of epithelioid MM are readily recognized by most pathologists: tubulopapillary, acinar (glandular), adenomatoid (also termed microglandular), and solid. Some epithelioid MMs have a distinctive feature consisting of clusters of tumor cells floating in pools of hyaluronic acid. Less commonly, tumor cells may be clear, deciduoid, signet ring, small cell, or rhabdoid or may have an adenoid cystic pattern.^{26,27,29–32,34} 

The tubulopapillary pattern consists of a mixture of papillary structures lined by bland flat, cuboidal, or polygonal cells with fibrovascular cores and glandlike tubules. Of note, a micropapillary pattern (without central fibrovascular core) should be classified as different from tubulopapillary, as the former correlates with a higher incidence of lymphatic invasion.²¹  The acinar pattern consists of elongated or branching glandlike lumina lined by relatively bland cuboidal cells. The adenomatoid pattern consists of bland, flat to cuboidal cells lining small glandlike structures.³⁵ 

The solid epithelioid MM consists of nests, cords, or sheets of round, oval, or polygonal cells with abundant eosinophilic cytoplasm and round, vesicular nuclei with prominent nucleoli. These cells resemble nonneoplastic, reactive mesothelial cells and the differential diagnosis may include reactive mesothelial hyperplasia, solid adenocarcinoma, and even squamous cell carcinoma owing to the abundant pink cytoplasm. The solid, poorly differentiated pattern consists of sheets and nests of relatively discohesive polygonal to round cells, with uniform nuclei. Lymphomas and poorly differentiated carcinomas enter into the differential diagnosis of solid, poorly differentiated MM. Recently, epithelioid mesotheliomas with marked nuclear pleomorphism in greater than 10% of the tumor have been shown to behave in similar fashion to sarcomatoid and biphasic variants, with a proposal that a “pleomorphic” variant be recognized as an adversely prognostic epithelioid pattern.^21,36 

The clear cell MM is composed of mesothelial cells with clear cytoplasm, which should be differentiated from clear cell renal cell carcinomas, clear cell carcinomas of the lung, clear cell melanoma, and other clear cell tumors that can metastasize to the pleura.^37–40 

The deciduoid MM is composed of sheets of large, round to polygonal cells with sharp cell borders, abundant glassy eosinophilic cytoplasm, and round vesicular nuclei with prominent nucleoli.

The adenoid cystic pattern consists of cribriform and tubular patterns separated by fibrous stroma, and the differential diagnosis includes adenoid cystic carcinoma in addition to adenocarcinoma. The signet ring and lipid-rich MMs consist of clusters or sheets of cells that contain cytoplasmic vacuoles; these rare tumors should be differentiated from metastatic signet ring cell adenocarcinoma and renal cell carcinoma, respectively.⁴¹  The extremely rare small cell MM consists of uniform small, round cells with bland nuclei and a high nuclear to cytoplasmic ratio.⁴²  The rhabdoid pattern is characterized by the presence of discohesive cells having abundant eosinophilic cytoplasm, an eccentric nucleus with a prominent nucleolus, and a rounded, eosinophilic cytoplasmic inclusion that sometimes causes nuclear indentation. The proportion of the rhabdoid component in these tumors ranges from 15% to 75%.⁴³ 

Secondary patterns of sarcomatoid MM may demonstrate anaplastic and giant cells with a differential diagnosis of high-grade sarcoma, osteosarcomatous areas with a differential diagnosis of osteosarcoma, or chondrosarcomatous areas with differential diagnosis of chondrosarcoma.^44–46 

The lymphohistiocytoid pattern (which may be better regarded as epithelioid subtype rather than sarcomatoid, since its prognosis is more like the former) consists of discohesive, atypical histiocytoid-appearing MM cells within an intense lymphoplasmacytic infiltrate. The differential diagnosis includes nonneoplastic inflammatory process, non-Hodgkin lymphoma, and Hodgkin lymphoma.^47,48 

Most desmoplastic MMs are sarcomatoid MMs, although occasional epithelioid desmoplastic MMs can occur. A paucicellular distribution of bland neoplastic spindle cells between bands of dense collagenous stroma that resemble pleural plaque is the distinguishing feature of desmoplastic MM. This type of MM may not be suspected unless frankly sarcomatoid areas of the tumor are found. This pattern is discussed further below. When prominent neoplastic giant cells or anaplastic cells are present (pleomorphic MM), pleomorphic carcinoma and other high-grade, poorly differentiated neoplasms metastatic to the pleura should be excluded.

Heterologous differentiation within a mesothelioma is a rare but well-established feature that occurs more frequently in sarcomatoid variants, although it can also be seen with biphasic and epithelioid morphologies. This most commonly takes the form of osteosarcomatous or chondrosarcomatous elements, although rarely, rhabdomyosarcomatous elements may be present.⁴⁹  One case showing angiosarcomatous differentiation has also recently been reported.⁵⁰  These elements are morphologically indistinguishable from the sarcomas themselves, and diagnosis is made on the basis of identifying the combined mesothelial elements, which usually predominate. In rare cases, mesothelial elements are in the minority and thorough sampling of any potential primary pleural sarcoma is recommended to exclude heterologous differentiation.

The morphology of peritoneal malignant mesothelioma (PMM) is similar to that of pleural MM in that there are epithelioid and sarcomatous types, with the former including the common tubulopapillary/papillary and solid histologic features. In the peritoneum, however, several site-specific issues are recognized.

While epithelioid and sarcomatous types can be seen in PMM, the incidence of biphasic tumors is lower than in pleural disease, and pure sarcomatous tumors are very rare.^51,52  As in pleural MM, the biphasic and sarcomatoid subgroups have a significantly poorer prognosis and are less amenable to treatment overall.^53,54  While definitions of pleural MM have proposed a minimum of 10% spindled growth for a biphasic designation, the less common occurrence of biphasic histologic appearance and the distinctly poorer prognosis of this group in PMM may make a minimum value less practical. It remains unclear whether identification of any component of malignant spindled histology portends a poor prognosis in PMM.⁵⁵ 

Multiple mesothelial-lined cysts, also known as benign multicystic mesothelioma, represent a rare but well-described entity that may enter the differential diagnosis of mesothelial neoplasia. This lesion is nearly always encountered in the peritoneum, although rare cases with pleural involvement have been described. These cystic proliferations are lined by bland mesothelial cells and lack stratification, papillation, or atypia. If defined in this fashion, this process does not metastasize but can recur.⁵⁶ 

Well-differentiated papillary mesothelioma (WDPM) is also an important subgroup much more frequently encountered in the peritoneum than in the pleura. These generally noninvasive papillary neoplasms are lined by bland mesothelial cells with low-grade nuclei. These nuclei are small, smoothly contoured, and do not contain nucleoli. Mitoses are rarely present. The combination of more-than-bland low-grade nuclei, architectural complexity or solid pattern, or overt invasion should be used to exclude WDPM in favor of papillary epithelioid malignant mesothelioma. In a recent series of WDPM in women,⁵⁷  1 of 26 patients had recurrent disease and none died of disease-related causes. No association with asbestos exposure was identified. The largest tumor in this series was 2.0 cm. Many cases had multifocality, however. Setting a size limit to the use of this diagnosis was proposed by these authors; it is clear, however, that bona fide cases can have a tumor size that exceeds 2.0 cm. It is acknowledged that bulky disease is one feature against WDPM. A discussion of size criteria remains an important open question, as the major concern in a larger or multifocal tumor is the undersampling or misclassification of a papillary epithelioid malignant mesothelioma as a WDPM. In summary, when narrowly defined by morphologic criteria, WDPM has an excellent prognosis, although recurrent disease can be problematic. Since the natural history of this subgroup is distinct from PMM, it is an important morphologic distinction from architecturally similar but more aggressive papillary epithelioid malignant mesotheliomas.^58,59 

The cytoplasmic vacuoles in adenocarcinomas frequently contain epithelial mucin highlighted by periodic acid–Schiff after digestion (PAS-D) and mucicarmine stains. Epithelial mucin can also be positive by Alcian blue but it is not digested by hyaluronidase. While it has been generally accepted that MMs do not show PAS-D–positive vacuoles, as seen in adenocarcinomas, there are rare published examples of epithelioid MM that show PAS-D positivity.⁶⁰  Mesothelial cells may have vacuoles containing hyaluronic acid, positive by Alcian blue and digestible by hyaluronidase. Mucicarmine may also stain hyaluronic acid in MM; thus, mucicarmine stain is not recommended for distinguishing MM from adenocarcinoma.

A definitive diagnosis of malignant mesothelioma requires a workup including immunohistochemistry and in some cases, histochemical stains for mucin. The role of immunohistochemistry varies depending on the histologic type of mesothelioma (epithelioid versus sarcomatoid), the location of the tumor (pleural versus peritoneal), and the type of tumor being considered in the differential diagnosis (adenocarcinoma, squamous cell carcinoma, malignant melanoma, epithelioid hemangioendothelioma). The immunohistochemical approach is also different depending on whether the tumor is sarcomatoid or epithelioid. Since biphasic mesotheliomas have an epithelioid component, the differential diagnosis is similar to that of epithelioid mesotheliomas.

Immunohistochemical staining for pancytokeratin is useful in the diagnosis of mesothelioma since virtually all epithelioid MMs and most sarcomatoid MMs will be positive. In a recent study,⁶¹  93% of sarcomatoid mesotheliomas exhibited immunoreactivity for cytokeratin (CK); this percentage may be even higher if a cocktail of keratins is used. Sarcomatoid MM with osteosarcomatous or chondrosarcomatous differentiation may be keratin negative. If an epithelioid malignant neoplasm causing diffuse pleural thickening is keratin negative with pancytokeratin immunostaining (using multiple keratins including AE1/AE3, CAM 5.2, and CK5/6), one should consider other possible differential diagnoses such as malignant melanoma, epithelioid hemangioendothelioma, or angiosarcoma (although some of these can be keratin positive), and malignant lymphoma. In this circumstance, it is recommended that a screening panel be performed to address these possibilities. Such a panel might include CD45, CD20, CD3, or CD30 for large cell lymphomas; S100 and HMB-45 for melanoma; and CD31 and CD34 for angiosarcoma and epithelioid hemangioendothelioma. Since D2-40 will stain epithelioid vascular tumors, it is not a good marker for this differential diagnosis. Further confirmatory staining may be useful if one or more of these screening markers show positivity. Ultrastructural studies may be of benefit in particularly difficult cases.

On occasion, a tumor may not stain with any marker. This lack of staining can be caused by a variety of reasons, including overfixation in formalin. Negative immunoreactivity may also occur in alcohol-fixed tissues if antigen retrieval is used; therefore, some knowledge about the fixative is important. If needed, vimentin may be used to assess immunoreactivity.

As the role of immunohistochemistry has evolved, it has become a standard to use panels of positive and negative antibodies that vary depending on the differential diagnosis. Since there is variability of staining between different antibody clones and between separate laboratories, no specific panel of antibodies is recommended. It is best for each laboratory to test staining conditions for the antibodies of choice with appropriate controls. If possible, one should choose antibodies with a sensitivity or specificity of at least 80%.

There is no absolute number of antibodies that can be recommended for the diagnosis of malignant mesothelioma. Workup can be done in stages. An initial workup could use 2 mesothelial markers and 2 markers for the other tumor under consideration on the basis of morphology (adenocarcinoma, squamous cell carcinoma). If the results are concordant, the diagnosis may be considered established. If they are discordant, a second stage, expanding the panel of antibodies, may be needed. The pattern of immunohistochemical staining is important with certain antibodies, such as calretinin, where both cytoplasmic and nuclear staining is required to support a diagnosis of mesothelioma, and Wilms tumor 1 (WT-1), which should be only nuclear. There is no standard for the percentage of tumor cells that should be positive, but some have used a 10% cutoff for membranous and cytoplasmic staining.

The differential diagnosis of epithelioid pleural mesothelioma is greatly facilitated by the use of immunohistochemistry. A relatively large number of markers that can assist in distinguishing epithelioid pleural mesothelioma from metastatic carcinoma originating either in the lung or in distant organs, such as the kidney, breast, or ovary, are currently available. Tables 5 and 6, respectively, list the markers that are currently useful in distinguishing epithelioid pleural mesotheliomas from lung adenocarcinomas, and those that discriminate between epithelioid mesotheliomas and squamous cell carcinomas. Since none of these markers are 100% specific, the IMIG recommends that at least 2 mesothelial and 2 carcinomas markers, in addition to cytokeratin (using a broad-spectrum anti-cytokeratin antibody), be included in any panel.¹  Based on their sensitivity and specificity, calretinin (Figure 11, A and B), CK5 or CK5/6 (Figure 12, A and B), WT-1 (Figure 13, A through C), and D2-40 (podoplanin) (Figure 14, A and B) are the best positive mesothelioma markers; and MOC-31 (Figure 15, A through C), Ber-EP4, carcinoembryonic antigen (CEA), and Lewis(y) antigen blood group 8 (BG8) are the best overall carcinoma markers.^62–64  Because of their high specificity for lung adenocarcinomas, thyroid transcription factor-1 (TTF-1) (Figure 16) and napsin A have an advantage over the other markers in that they can be used to confirm the pulmonary origin of an adenocarcinoma. MOC-31, Ber-EP4, CEA, BG8 (Figure 17), and p63 are regarded as the best positive carcinoma markers for differentiating between epithelioid mesotheliomas and squamous cell carcinomas because they are commonly expressed in the latter and are usually absent in the former.⁶⁵  p63 has an advantage over the other 4 markers in that, in addition to being strongly and invariably expressed in squamous cell carcinomas, while it is absent in mesotheliomas, it may also assist in distinguishing squamous cell carcinomas from pulmonary adenocarcinomas. Because WT-1 is expressed in most epithelioid mesotheliomas, but absent in squamous cell carcinomas, it is the best positive mesothelioma marker for discriminating between these malignancies. Calretinin is not as useful in this scenario since it often shows positivity in squamous cell carcinomas.

Other carcinomas that metastasize to the pleura, and which can potentially be confused with mesothelioma, are those that originate in the ovary and fallopian tube (discussed later), breast, kidney, and gastrointestinal tract. Since most breast carcinomas express estrogen receptor, gross cystic disease fluid protein-15, or mammaglobin, immunostaining for these markers can be very useful in distinguishing mesothelioma from a metastatic breast carcinoma. Markers useful in differentiating mesothelioma from metastatic renal cell carcinoma are given in Table 7. Because of their sensitivity and specificity, calretinin, D2-40 (podoplanin), and cytokeratin 5/6 are the best positive mesothelioma markers.⁶⁶  Among the carcinoma markers, PAX8 or PAX2 are the most useful as they are expressed in most renal cell carcinomas (Figure 18),⁶⁷  but not in mesotheliomas.⁶⁸  Renal cell carcinoma marker and CD15 can also be useful, but the sensitivity and specificity of these markers for renal cell carcinomas is significantly lower than those of PAX8 or PAX2.

Diffuse malignancies of the peritoneum include PMM and secondary peritoneal carcinomatosis in the clinical, imaging, and gross pathologic differential diagnosis in many cases. In pleural disease, pseudomesotheliomatous carcinoma (defined as a carcinoma that grows along pleura encasing the lung) is most often from an adenocarcinoma of pulmonary origin, while peritoneal carcinomatosis can be of ovarian, fallopian tube (previously considered as primary peritoneal carcinomas), gastric, pancreatic, colonic, and more rarely, breast origin.^51,69  Therefore, immunohistochemistry panels have to be adjusted accordingly.

Most studies have focused on differentiating PMM from papillary serous carcinoma (PSC) and these are summarized in Table 8. There have been fewer data directly comparing the profile of PMM to pancreatic, gastric, and colon carcinoma. The markers useful in female patients include calretinin, and possibly D2-40 (which can also be positive in some cases of PSC), for positive markers in PMM; and MOC-31, BG8, and with less specificity, Ber-EP4, for positive adenocarcinoma markers. While specific, B72.3 staining may be too focal in many PSC cases, although a positive result is very useful. The high frequency of reactivity for the mesothelioma markers CK5/6 and WT-1 in PSC and the less frequent staining for CEA in PSC limits the ability of those markers to discriminate between these entities. Carcinoembryonic antigen may also be useful in the setting when PSC is not in the differential diagnosis. Although h-caldesmon has been reported to be highly useful as a mesothelial marker,⁷⁰  other studies⁶⁸  have not shown this. A strongly positive result for estrogen receptor may be helpful in difficult cases, as would a positive result for progesterone receptor. A very useful marker to address the problem of tumors of müllerian origin in women and tumors of renal origin in all patients is PAX8.^68,71  PAX8 is a transcription factor involved in the development of thyroid, kidney, and müllerian system. While focal or weak staining can be seen in a small number of mesotheliomas, a high percentage of ovarian, tubal, endometrial, and renal tumors show immunoreactivity that is frequently diffuse and intense. This marker is very promising when added to a panel to differentiate abdominal malignant mesothelioma from carcinoma.

In male patients, WT-1 (nuclear staining) and D2-40 are useful markers in addition to calretinin for MM, and for nonserous adenocarcinoma, B72.3, MOC31, BG8, and Ber-EP4 all have high sensitivity and specificity.

The criteria for distinguishing reactive fibrous pleurisy from sarcomatoid mesothelioma have been well characterized and are summarized above.

An immunohistochemical panel that can be useful for the initial evaluation of a sarcomatoid tumor involving the pleura should include cytokeratins, calretinin, and D2-40. Multiple cytokeratin antibodies including AE1/3, CAM 5.2 (or CK18), and CK7 should be used, as cytokeratin expression can be focal, weak, and/or variable.^72,73  Other positive markers that are used in the evaluation of epithelioid mesothelioma, such as WT-1 and CK5/6, as well as adenocarcinoma markers, such as Ber-EP4, CEA, and MOC-31, do not provide much added utility in sarcomatoid tumors. D2-40 and calretinin have been the 2 positive mesothelial markers most consistently expressed in sarcomatoid mesotheliomas in a variable percentage of cases.^74,75  False positives can occur by the misinterpretation of positive D2-40 reactivity within benign entrapped lymphatics or reactive mesothelial elements. A recent study⁶¹  reported the presence of usually focal calretinin immunoreactivity labeling fewer than 10% of tumor cells in 31% of sarcomatoid mesotheliomas.

A histologically malignant sarcomatoid tumor that is strongly and diffusely cytokeratin positive usually limits the differential diagnosis to sarcomatoid mesothelioma, sarcomatoid carcinoma, and, on occasion, synovial sarcoma or metastatic sarcomatoid renal cell carcinoma. Although synovial sarcomas of the pleura (or primary pulmonary synovial sarcomas involving the pleura) usually present as localized solid tumors, they can present with diffuse pleural thickening that is similar to malignant mesothelioma. The diagnosis of synovial sarcoma should be considered when there is a highly cellular neoplasm with very little cytoplasm in tumor cells, resulting in overlapping nuclei with the presence of focal hemangiopericytoma-like blood vessels and limited keratin expression. The diagnosis can be confirmed by molecular testing for its distinctive X:18 translocation in formalin-fixed, paraffin-embedded tissue. Unless there is convincing calretinin and D2-40 positivity, it is difficult to separate out the spindled cell component of a partially sampled sarcomatoid carcinoma from sarcomatoid mesothelioma. Heterologous elements may be present in both tumors. A possible distinguishing feature is when the tumor has areas where the malignant cells are infiltrating through densely collagenized fibrosis (as is characteristic of desmoplastic mesothelioma). This pattern is quite typical of malignant mesothelioma and favors that diagnosis, although ultimately, in this instance, the diagnosis may have to incorporate other gross and clinical features. In some cases, especially with limited biopsy material, it may be difficult to distinguish metastatic sarcomatoid carcinoma from sarcomatoid mesothelioma. Carcinoma markers such as CEA and TTF-1 (for lung) can be tried. Sarcomatoid renal cell carcinoma can metastasize to the pleura and grow like a mesothelioma, producing a pseudomesotheliomatous sarcomatoid-type pattern. Differential cytokeratin positivity profiles, other than CK5/6, have not been reported to date in the differential diagnosis of these 2 tumors. CK5/6 shows negativity in sarcomatoid renal cell carcinomas, but the low sensitivity of CK5/6 as a marker in sarcomatoid mesothelioma greatly limits its utility.⁶⁶  Calretinin and D2-40 positivity have not been extensively studied in sarcomatoid renal cell carcinomas. One series reported calretinin negativity in all four sarcomatoid renal cell carcinomas tested but at this point, it would be prudent to incorporate additional gross and clinical correlation.⁶⁶ 

A histologically malignant sarcomatoid tumor that is either focally cytokeratin positive or cytokeratin negative should be cautiously interpreted, and a diagnosis of mesothelioma very carefully considered. Focal cytokeratin positivity has been reported in many different types of sarcomas. It is also possible that the focal cytokeratin positivity represents entrapment of benign pleural elements. If the results from the initial round of cytokeratins prove to be negative or there is only focal cytokeratin positivity, additional blocks should be selected and stained, and cytokeratin antigen retrieval techniques, as well as antibody source and dilutions, should be reviewed. A vimentin stain is useful in assessing the general antigenic integrity of the tissue. Particularly in the absence of convincing cytokeratin positivity, calretinin and/or D2-40 positivity alone should not be interpreted as evidence of mesothelial differentiation. These markers show variable positivity in many different types of sarcomas, and other immunohistochemical markers should be added at this point. The expanded differential diagnosis might include other sarcomas (epithelioid hemangioendothelioma/angiosarcoma, synovial sarcoma, liposarcoma, myogenic or neurogenic tumors), malignant solitary fibrous tumor, melanoma, and lymphoma. The marker panel should be expanded accordingly to include antibodies such as CD31, CD34, desmin, myoglobin, S100, and CD45. It should be noted that some muscle markers are often positive, at least focally, and on occasion more diffusely, in sarcomatoid mesotheliomas.⁷⁶  These markers include muscle-specific actin and α smooth muscle actin. In contrast to reactive mesothelial cells, desmin positivity in pure sarcomatoid mesotheliomas is quite rare.^76,77  After extensive workup and with appropriate clinical and radiologic features, cytokeratin-negative sarcomatoid mesotheliomas have been published as a diagnosis of exclusion.^72,78,79 

Key molecular alterations in pathogenesis of malignant mesothelioma have been known for decades, but their potential diagnostic and prognostic implications have only recently been more extensively investigated.⁸⁰  One of the most common genetic alterations in mesothelioma is the homozygous deletion of the 9p21 locus within a cluster of genes that includes cyclin-dependent kinase inhibitor 2A (CDKN2A), CDKN2B, and methylthioadenosine phosphorylase (MTAP).^81–85  Several cytogenetic and molecular studies have reported p16/CDKN2A deletions in up to 80% of primary pleural mesotheliomas, depending on the histologic subtype (90%–100% of sarcomatoid mesothelioma, 70% of epithelioid and mixed types). In contrast, this deletion occurs in approximately 25% of peritoneal mesotheliomas.^18,85,86  Besides homozygous deletion, point mutations and DNA methylation occur less frequently at the same genetic locus.⁸³  P16/CDKN2A is present in all normal cells and is essential for normal cell cycle control, and therefore its loss may be a helpful marker of malignancy. It has been demonstrated that deletions of p16/CDKN2A occur in malignant mesotheliomas only, whereas point mutations and DNA methylation may occur in benign mesothelial cells as well.⁸⁷  Therefore, the detection of deletion can be a useful approach for distinguishing benign from malignant mesothelial proliferations. It should be emphasized that this technique is not useful for distinguishing malignant mesothelioma from adenocarcinoma (as discussed below).

Various methods, including polymerase chain reaction–based techniques and FISH, have been used in detection of deletions. The FISH assay can be performed by using a commercially available dual-color FISH probe (Abbott Molecular, Des Plaines, Illinois). It can be reliably performed on archival paraffin-embedded tissue and is relatively less expensive than other molecular assays. Another advantage of this technique over polymerase chain reaction–based assays is the ability to identify homozygous and hemizygous deletions. Furthermore, different tumor areas can be simultaneously analyzed and visualized. The FISH technique for detection of 9p21 deletions has been shown to be a very powerful technique for confirming the diagnosis of malignant mesothelioma in effusion and formalin-fixed, paraffin-embedded tissue specimens (Figure 19, A and B).^{19,20,86,88,89} 

The diagnosis of atypical mesothelial proliferation is more common in cytologic specimens than in surgical specimens because the diagnosis of mesothelioma can be more challenging in cytologic specimens because of the inability to evaluate for tissue invasion and of the numerous cytomorphologic mimics of mesothelioma, including reactive mesothelial proliferations. In the diagnosis of MM in effusion cytology across all cytologic categories, studies showed an overall sensitivity of p16 FISH between 56% and 79% with a positive predictive value of 100%. FISH p16 also showed better sensitivity and specificity than GLUT-1 immunohistochemical marker in cytology specimens.²⁰  The main challenge in the assessment of p16 deletion by FISH in cytology specimens when cell block is available is the presence of admixed reactive mesothelial cells that could be morphologically indistinguishable from malignant mesothelial cells and could potentially lead to false-negative FISH results.

Although studies showed statistically proven good correlation between p16 deletion and lack of p16 protein expression, there is a subset of cases where p16 protein expression would be maintained despite the presence of p16 gene deletion and vice versa. This could be explained by the type of antibody, assay conditions, preanalytic variables, and interpretation criteria. Therefore, immunohistochemical assessment for loss of p16 protein expression would be unreliable and should not be used as a surrogate method for detection of p16 deletion.⁸⁶ 

Homozygous deletion of the p16 gene can be used not only as diagnostic, but also as a prognostic marker. It has been demonstrated that the presence of p16 homozygous deletion correlates with a shorter survival in patients with malignant mesotheliomas.^85,90,91  There is also a correlation between p16 protein loss, as demonstrated by immunohistochemistry, and a poor prognosis with increased risk of death in peritoneal mesothelioma, but the association is not as strong.^55,90  There are no molecular markers to help distinguish malignant mesotheliomas from carcinomas or sarcomas on formalin-fixed, paraffin embedded tissue. Genetic alterations of 9p are one of the most frequent events in other tumor types including non–small cell carcinomas of the lung, melanoma, and sarcomas; therefore, deletion cannot be used to differentiate these neoplasms from malignant mesotheliomas.^92–94  However, detecting t(X:18) is most useful in the differential diagnosis of synovial sarcoma. Begueret et al⁹⁵  have confirmed the presence of this translocation in 90% of purely sarcomatoid primary synovial sarcomas of the pleura, while this translocation has never been detected in malignant mesothelioma.⁹⁶ 

DNA methylation profiles, microRNA dysregulation, and BAP1 mutations are being studied, which are likely to yield important results in understanding pathogenesis and developing targeted therapy for MM, but are currently not used for diagnosis.

The electron microscopic features of malignant mesothelioma are well described.^30,97  There is no single ultrastructural feature that is diagnostic of mesothelioma, but rather, a combination of several features may be diagnostically useful. For epithelioid mesotheliomas these include very long, thin apical microvilli that do not have a glycocalyx, as opposed to the generally shorter microvilli of adenocarcinomas that usually do have a glycocalyx; perinuclear tonofilament bundles; the presence of basal lamina; and long desmosomes.

It should be emphasized that the role of electron microscopy in this setting is very restricted because most tumors that show the ultrastructural features described above are epithelial mesotheliomas for which light microscopy with immunohistochemistry is vastly faster and often cheaper (and more widely available) in establishing the correct diagnosis. Sarcomatous mesotheliomas for the most part do not show specific ultrastructural features, and tumors that are poorly differentiated by light microscopy and do not demonstrate a typical pattern of immunohistochemical staining usually lack specific features by electron microscopy as well.^98,99  Occasionally, electron microscopy is useful in establishing the correct diagnosis when the immunohistochemical results are equivocal or further support of a diagnosis of either MM or serous carcinoma is needed.⁶⁵  Formalin-fixed material retrieved from a paraffin block may be satisfactory, since microvilli and tonofilament bundles tend to be preserved.

Because there is an association of asbestos exposure and the development of malignant mesothelioma, many pathologists may adopt the position that a history of asbestos exposure makes a tumor more likely to be a mesothelioma, and, conversely, in the absence of such a history, they are reluctant to diagnose mesothelioma. However, the history of exposure to asbestos or the absence of such a history is not useful to the pathologist in making a diagnosis of mesothelioma. The situation is analogous to that of lung cancer: although most lung cancers occur in cigarette smokers, no one would hesitate to diagnose a lung cancer if told that the patient was a nonsmoker. For mesothelioma a similar scenario applies: the diagnosis is based on clinical, radiologic, and, ultimately, pathologic features, and the issue of asbestos exposure is irrelevant. Attribution of cause is a separate issue.

Like many tumors with a papillary architecture, mesotheliomas may occasionally contain psammoma bodies. While the presence of psammoma bodies might suggest certain carcinomas (such as papillary carcinoma in the thyroid and serous papillary tumors of the female genital tract), their presence is not a diagnostic aid in either confirming or excluding a diagnosis of mesothelioma.

While the presence of mucin has been classically used to support a diagnosis of adenocarcinoma over mesothelioma, mucin positivity may be encountered in mesotheliomas. With Alcian blue and colloidal iron staining, mesotheliomas that may contain hyaluronate droplets show a positive reaction; this material is removed with hyaluronidase pretreatment. The spaces containing the positive material tend to be quite large and not typical of the intracellular mucin droplets seen in adenocarcinoma. Because hyaluronidase-sensitive material may be encountered in mesotheliomas, PAS-D stains have been recommended for neutral (“epithelial”) mucins, since this finding is almost entirely restricted to adenocarcinoma. Rarely, otherwise typical mesotheliomas may contain mucicarmine-positive, PAS-D–positive, and Alcian blue–positive droplets that are resistant to hyaluronidase. Ultrastructural evaluation in the small series reported by Hammar⁶⁰  suggested that this positivity was related to crystallized proteoglycans.

Faint mucicarmine positivity is common in the vacuoles of mesotheliomas and should not be considered a feature diagnostic of adenocarcinoma.

Simian virus 40 has been proposed by some as an etiologic agent in some mesotheliomas. While this proposal remains controversial it is clear that the presence or absence of exposure to simian virus 40 is not a criterion in confirming or excluding the diagnosis of mesothelioma.³⁰ 

The first “port of call” for the histologic diagnosis of malignant mesothelioma is the morphology. Immunohistochemical stains are important for confirmation of the diagnosis, but they should not be used to force a tumor into the diagnosis of mesothelioma when it does not look like a mesothelioma on hematoxylin-eosin; neither should they be performed automatically or blindly without considering several factors. As stated previously, the major determinants of which panel to use are (1) the location of the tumor—it will vary as to whether it is pleural, peritoneal, or another serosal surface; (2) the phenotypic problem—benign versus malignant, epithelioid, spindle, biphasic, small cell, pleomorphic, and (3) the experience of the laboratory. A laboratory using immunohistochemical stains should be performing them frequently, have well-established protocols, and should have an appreciation of their sensitivities and specificities with respect to the various morphologic problems. There is no single utopian immunohistochemical panel to cover all diagnostic “mesothelial” problems.

One of the problems in comparing the results of particular antibodies from different studies is a lack of standardization of immunohistochemical procedures. This can result in conflicting results for sensitivity and specificity for various antibodies. The study of King et al⁴  (2006) tabulates the data for antibody clone, manufacturer, dilution, and antigen retrieval methods for 5 antibodies used in separating benign and malignant mesothelial proliferations in 13 studies. It illustrates the wide variability between the various studies. Before using an antibody for diagnosis, a laboratory should have carried out an extensive workup to find the ideal conditions for routine use.⁴ 

The type of pathologic sample may affect results. For example, tiny needle biopsy samples may show crush artifact and false-positive immunostaining with various antibodies. Also, the edges of biopsy specimens may show artifactual positive immunostaining. There may also be variation in interpretation of what is positive, illustrated by the fact that some laboratories will only consider calretinin as showing positivity when there is nuclear staining, whereas a minority will consider cytoplasmic staining as showing positivity. This can significantly affect sensitivity and specificity.

Another problem associated with immunohistochemistry may be putting too much emphasis on focal immunopositivity. We would suggest that weak or focal staining of fewer than 10% of the cells should be considered as being negative when interpreting a panel of stains. Also, one can observe positive immunostaining with mesothelial markers in reactive proliferations of submesothelial fibroblasts in the vicinity of nonmesothelial tumors and inflammatory pleural diseases—it is important not to diagnose these as mesotheliomas. In contrast, mesotheliomas may invade the underlying lung, and entrapped pulmonary epithelial cells may show positive immunostaining with epithelial markers. Careful correlation with the hematoxylin-eosin sections is necessary to avoid misinterpretation.

It is also important to know the full range of cell types an individual marker may stain. For example, WT-1 and D2-40 (podoplanin) show positivity in endothelial cells, which should not be misinterpreted as positive tumor staining in small, crushed biopsy samples in particular. Similarly, “mesothelial markers” may show positivity in tumors other than mesothelioma. For example, WT-1 may show positivity in ovarian serous tumors and melanoma, calretinin in synovial sarcoma and some germ cell tumors, and CK 5/6 in squamous carcinomas. As such, the significance of positive staining in a single marker should be interpreted within the context of the totality of immunohistochemical, morphologic, and clinical findings.

Another pitfall leading to misdiagnosis may result from “false” invasion, which can apply to the pleura or chest wall fat. Inflammatory pleural processes may result in mesothelial cells lying quite deeply within the pleura (as a result of entrapment in granulation tissue), but these are usually parallel with the pleural surface. Sometimes one sees tubular collections of reactive mesothelial cells, which are lined up parallel to the pleural surface. These do not connote malignancy. Sometimes a section taken parallel to the pleural surface may give a false impression of a full-thickness mesothelial proliferation. Organizing pleuritis may result in a “fake fat” phenomenon whereby a greatly thickened fibrotic paucicellular pleura is associated with circular fatlike spaces and with cytokeratin-positive spindle cells running between these fatlike spaces (Figure 7). However, they are parallel to the pleural surface and vimentin stain shows that there is no cellular lining to the spaces. On the other hand, desmoplastic mesothelioma usually shows a downward rather than horizontal growth pattern of the keratin-positive spindle cells (Figure 5, B).¹¹ 

Mesothelioma review panels have been functioning since the 1960s. These panels have served as a referral source for pathologists facing diagnostic problems and, more recently, to confirm diagnoses for treatment trials. In North America there is the US and Canadian Mesothelioma Panel (overseen by A. C.) and there are several in Europe, with the best known and most productive being the French Mesopanel (overseen by F. G.-S.). The diagnosis of mesothelioma has been considered to be difficult, although the application of immunohistochemical staining has made the diagnosis more reliable. In a 1991 report from the US and Canadian Mesothelioma Panel, only 70.5% of 200 cases had a three-fourths majority agreement from the panel. It should be emphasized that these represented referral cases and by their nature were already difficult. In a more recent report from the same panel,²⁸  agreement among panel members as to benign versus malignant was 78% for a group of 217 cases.

This article gives broad guidelines for making the diagnosis of MM, which although a rare tumor, has a grave prognosis and invariably has medicolegal implications. The salient recommendations are use of histologic features and immunohistochemical panel in distinguishing benign from malignant mesothelial proliferations and the use of molecular assays, such as homozygous p16 deletion, in challenging cases; on biopsy, subtyping should be done, but assigning a further pattern is often not possible; there is limited usefulness of cytology, histochemical stains, and electron microscopy; panels of antibodies need to be used according to the differential diagnosis in each case; in the typical case in which all features are concordant, 2 mesothelioma markers and 2 carcinoma markers may be adequate to make a diagnosis, but when there are discordant findings, additional markers should be used. The pathologist should always take the clinical, radiologic, and pathologic features into consideration and get expert second opinion in difficult cases, as necessary.