As part of the International Mesothelioma Interest Group

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1 Review Articles Guidelines for Pathologic Diagnosis of Malignant Mesothelioma 2012 Update of the Consensus Statement from the International Mesothelioma Interest Group Aliya N. Husain, MD; Thomas Colby, MD; Nelson Ordonez, MD; Thomas Krausz, MD; Richard Attanoos, MB, BS; Mary Beth Beasley, MD; Alain C. Borczuk, MD; Kelly Butnor, MD; Philip T. Cagle, MD; Lucian R. Chirieac, MD; Andrew Churg, MD; Sanja Dacic, MD, PhD; Armando Fraire, MD; Francoise Galateau-Salle, MD; Allen Gibbs, MD; Allen Gown, MD; Samuel Hammar, MD; Leslie Litzky, MD; Alberto M. Marchevsky, MD; Andrew G. Nicholson, DM; Victor Roggli, MD; William D. Travis, MD; Mark Wick, MD Accepted for publication June 13, Published as an Early Online Release August 28, From the Department of Pathology, University of Chicago, Chicago, Illinois (Drs Husain and Krausz); the Department of Pathology, Mayo Clinic Arizona, Scottsdale (Dr Colby); the Department of Pathology, MD Anderson Cancer Center, Houston, Texas (Dr Ordonez); the Department of Cellular Pathology, University Hospital Llandough, Cardiff, United Kingdom (Mr Attanoos); the Department of Pathology, Mount Sinai Hospital, New York, New York (Dr Beasley); the Department of Pathology, Columbia University Medical Center, New York, New York (Dr Borczuk); the Department of Pathology, University of Vermont College of Medicine, Burlington (Dr Butnor); the Department of Pathology & Genomic Medicine, The Methodist Hospital, Houston, Texas (Dr Cagle); the Department of Pathology, Brigham and Women s Hospital, Boston, Massachusetts (Dr Chirieac); the Department of Pathology, University of British Columbia, Vancouver, Canada (Dr Churg); the Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (Dr Dacic); the Department of Pathology, University of Massachusetts, Worcester (Dr Fraire); Laboratoire d Anatomie Pathologique, Caen- Cedex, France (Dr Galateau-Salle); the Department of Histopathology, Llandough Hospital, Penarth, South Glamorgan, United Kingdom (Dr Gibbs); PhenoPath Laboratories, Seattle, Washington (Dr Gown); Diagnostic Specialties Laboratory, Bremerton, Washington (Dr Hammar); the Department of Pathology & Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia (Dr Litzky); the Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, California (Dr Marchevsky); the Department of Histopathology, Royal Brompton Hospital, London, United Kingdom (Mr Nicholson); the Department of Pathology, Duke University Medical Center, Durham, North Carolina (Dr Roggli); the Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York (Dr Travis); and the Department of Pathology, University of Virginia Medical Center, Charlottesville (Dr Wick). Dr Churg serves as a consultant to law firms in asbestos litigations. Dr Gibbs undertakes medicolegal work related to mesothelioma. Dr Roggli testifies as an expert witness in asbestos litigations. The other authors have no relevant financial interest in the products or companies described in this article. Presented at the Pulmonary Pathology Society companion meeting in conjunction with the United States and Canadian Academy of Pathology annual meeting; March 17, 2012; Vancouver, British Columbia, Canada. Reprints: Aliya N. Husain, MD, Department of Pathology, MC6101, University of Chicago Medical Center, 5841 S Maryland Ave, Room S-627, Chicago, IL ( aliya.husain@ uchospitals.edu. Context. Malignant mesothelioma (MM) is an uncommon tumor that can be difficult to diagnose. Objective. To provide updated practical guidelines for the pathologic diagnosis of MM. Data Sources. Pathologists involved in the International Mesothelioma Interest Group and others with an interest in the field contributed to this update. Reference material includes peer-reviewed publications and textbooks. Conclusions. There was consensus opinion regarding (1) distinction of benign from malignant mesothelial proliferations (both epithelioid and spindle cell lesions), (2) cytologic diagnosis of MM, (3) key histologic features of pleural and peritoneal MM, (4) use of histochemical and immunohistochemical stains in the diagnosis and differential diagnosis of MM, (5) differentiation of epithelioid MM from various carcinomas (lung, breast, ovarian, and colonic adenocarcinomas, and squamous cell and renal cell carcinomas), (6) diagnosis of sarcomatoid mesothelioma, (7) use of molecular markers in the diagnosis of MM, (8) electron microscopy in the diagnosis of MM, and (9) some caveats and pitfalls in the diagnosis of MM. Immunohistochemical panels are integral to the diagnosis of MM, but the exact makeup of panels used is dependent on the differential diagnosis and on the antibodies available in a given laboratory. Immunohistochemical panels should contain both positive and negative markers. It is recommended that immunohistochemical markers have either sensitivity or specificity greater than 80% for the lesions in question. Interpretation of positivity generally should take into account the localization of the stain (eg, nuclear versus cytoplasmic) and the percentage of cells staining (.10% is suggested for cytoplasmic membranous markers). These guidelines are meant to be a practical reference for the pathologist. (Arch Pathol Lab Med. 2013;137: ; doi: / arpa oa) As part of the International Mesothelioma Interest Group (IMIG) biennial meeting held in Chicago (October 2006), there was a pathology half-day workshop that Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al 647

2 included invited lecturers and an open forum on the pathologic diagnosis of malignant mesothelioma (MM). The discussion focused on practical diagnostic guidelines meant to be a reference for the pathologist, rather than a mandate or review of the literature. With input from other pathologists who could not attend the meeting, an article titled Guidelines for Pathologic Diagnosis of Malignant Mesothelioma was published in This article represents an update by the original contributors and includes contributions from additional pathologists with expertise in this area. GENERAL RECOMMENDATIONS 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. BENIGN VERSUS MALIGNANT MESOTHELIAL CELL PROLIFERATIONS 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. Reactive Mesothelial Hyperplasia Versus Epithelioid MM 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. Fibrous Pleurisy Versus Desmoplastic Variant of Sarcomatoid Mesothelioma 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: 648 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al

3 Figure 1. Reactive mesothelial hyperplasia within fibrous tissue, mimicking invasion (hematoxylin-eosin, original magnification 3100). Figure 2. Epithelioid malignant mesothelioma invading fat ((hematoxylin-eosin, original magnification 3100). Figure 3. A through D, Glucose transporter 1 (GLUT-1) staining of mesothelial lesions. A, Malignant mesothelioma with strong immunoreactivity for GLUT-1. B, Mesothelial hyperplasia negative for GLUT-1. As expected, staining for red blood cells is strongly positive. C, Well-differentiated papillary mesothelioma is negative for GLUT-1. D, Sarcomatoid mesothelioma with positive staining for GLUT-1 (original magnifications 3400 [A and D]; original magnifications 3200 [B and C]). Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al 649

4 Table 1. Reactive Mesothelial Hyperplasia Versus Mesothelioma Mesothelial Hyperplasia Mesothelioma Absence of stromal invasion Stromal invasion usually apparent (beware of entrapment and en face cuts) (highlight with pancytokeratin staining) Cellularity may be prominent but is confined to the Dense cellularity including cells surrounded by stroma mesothelial surface/pleural space and not in the stroma Simple papillae; single cell layers Complex papillae; tubules, cellular stratification Loose sheets of cells without stroma Cells surrounded by stroma ( bulky tumor may involve the mesothelial space without obvious invasion) Necrosis rare Necrosis present (occasionally) Inflammation common Inflammation usually minimal Uniformity of growth Expansile nodules; disorganized growth (highlighted with cytokeratin staining) (highlighted with cytokeratin staining) EMA, p53, GLUT-1, and IMP3 usually stain negatively EMA, p53, GLUT-1, and IMP3 often stain positively Desmin often stains positively Desmin often stains negatively Usually Not Useful Mitotic activity Cytologic atypia Abbreviations: EMA, epithelial membrane antigen; GLUT-1, glucose transporter 1; IMP3, insulin-like growth factor II messenger RNA binding protein 3. 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 al 11 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 cytokeratinpositive 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, Table 2. Immunohistochemistry to Separate Reactive Mesothelial Proliferations from Mesothelioma a Antibody Reactive Mesothelium, No. (% Positive) Mesothelioma, No. (% Positive) Desmin 34/40 (85) 6/60 (10) EMA 8/40 (20) 48/60 (80) p53 0/40 (0) 27/60 (45) GLUT-1 5/150 (3) 103/153 (67) IMP3 0/64 (0) 33/45 (73) Abbreviations: EMA, epithelial membrane antigen; GLUT-1, glucose transporter 1; IMP3, insulin-like growth factor II messenger RNA binding protein 3. a Data derived from Kato et al, 5 Acurio et al, 6 Shi et al, 8 Monaco et al, 20 and Attanoos et al. 78 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. CYTOLOGIC DIAGNOSIS OF MALIGNANT MESOTHELIOMA 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, thinlayer) 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 demonstrat- 650 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al

5 Figure 4. Epithelioid malignant mesothelioma with strong cytoplasmic staining for insulin-like growth factor II messenger RNA binding protein 3 (IMP3) (original magnification 3400). Figure 5. A and B, Desmoplastic mesothelioma. A, Proliferation of bland-appearing spindle cells with haphazard growth pattern. B, Keratin staining highlights infiltration into fat (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B]). Figure 6. A and B, Fake fat in a pleural biopsy sample from a patient with effusion and fibrosis (hematoxylin-eosin, original magnifications 340 [A] and 3100 [B]). Figure 7. Staining for keratin AE1/AE3 showing horizontal keratin-positive reactive spindle cells around fake fat (see Figure 5, B, for comparison with adipose tissue) (original magnification 3100). Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al 651

6 Table 3. Fibrous Pleurisy Versus Desmoplastic Mesothelioma Fibrous Pleurisy Desmoplastic Mesothelioma Storiform pattern not prominent Absence of stromal invasion Necrosis, if present, is at the surface (where there is often associated acute inflammation) Uniform thickness of the process Hypercellularity at the surface with maturation and decrease in cellularity with depth (so-called zonation) Perpendicularly oriented vessels Storiform pattern often prominent Stromal invasion present (highlight with pancytokeratin staining) Bland necrosis of paucicellular collagenized tissue Disorganized growth with uneven thickness, expansile nodules, and abrupt changes in cellularity Lack of maturation from the surface to depths of the process Paucity of vessels, without orientation Usually Not Useful Cellularity Atypia (unless severe) Mitotic activity unless numerous atypical mitotic figures ing homozygous deletion of the p16 gene in about 70% of mesothelial proliferations, are particularly promising, as reported specificity is 100% However, emerging data indicate that subtyping of epithelioid mesothelioma according to morphologic features and nuclear grade 21 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, fineneedle 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). HISTOLOGIC FEATURES OF MM 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 MM 23 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 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 652 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al

7 Figure 8. A through F, S100, laminin, and collagen IV staining is negative in fake fat (A through C) and positive in true fat (D through F) (original magnifications 3200 [A through F]). 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 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al 653

8 Figure 9. A and B, Reactive fibrous pleuritis. A, There is uniformity of growth and thickness of the reactive process. B, Perpendicular, uniformly spaced thin-walled vessels with fibrin on one surface and progressive maturation of fibrous tissue in deeper part (hematoxylin-eosin, original magnifications 340 [A] and 3100 [B]). Figure 10. A through D, Cytologic features of malignant mesothelioma (MM). A, Numerous large clumps of cells are present in effusion of MM. B, The clumps have a berrylike external contour. C, Multiple binucleated cells are seen. D, Cell block also shows frequent clumps and can be very useful in performing special stains (Papanicolaou, original magnifications 340 [A], 3200 [B], and 3400 [C]; hematoxylin-eosin, original magnification 3200 [D]). 654 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al

9 Table 4. Histologic Subtypes and Patterns a of Malignant Mesothelioma Epithelioid mesothelioma Tubulopapillary Micropapillary Trabecular Acinar Adenomatoid Solid Clear cell Deciduoid Adenoid cystic Signet ring cell Small cell Rhabdoid Pleomorphic Sarcomatoid mesothelioma Conventional, spindle cell Desmoplastic Heterologous differentiation (osteosarcomatous, chondrosarcomatous, etc) Lymphohistiocytoid (may also be classified as epithelioid) Biphasic/mixed a Subtype must be given in the diagnosis, but histologic pattern, epithelioid or sarcomatous, may be described in a comment or microscopic description. 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 aproposalthata 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 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 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. MORPHOLOGIC FEATURES RELATED TO PERITONEAL MM 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 sitespecific issues are recognized. 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10 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 welldescribed 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 HISTOCHEMICAL STAINING IN MM 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. IMMUNOHISTOCHEMICAL STAINING IN MM 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 656 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al

11 Table 5. Immunohistochemical Markers Used in the Differential Diagnosis Between Epithelioid Pleural Mesothelioma and Lung Adenocarcinoma Marker Current Value/Comments Epithelioid mesothelioma (positive mesothelioma markers) Calretinin Very useful. It can be demonstrated in nearly all epithelioid mesotheliomas when antibodies to human recombinant calretinin are used. The staining is often strong and diffuse, and both nuclear and cytoplasmic. Five percent to 10% of lung adenocarcinomas are positive, but the staining is usually focal. Cytokeratin 5 or 5/6 Very useful. It is expressed in 75% to 100% of the mesotheliomas. Approximately 2% to 20% of lung adenocarcinomas can be focally positive. WT-1 Very useful. Approximately 70% to 95% of the mesotheliomas show nuclear positivity. Lung adenocarcinomas are negative. D2-40 (podoplanin) Very useful. Approximately 90% to 100% of mesotheliomas show positivity along the cell membranes. Up to 15% of lung adenocarcinomas are focally positive. Lung adenocarcinoma (positive carcinoma markers) MOC-31 Very useful. Approximately 95% to 100% of lung adenocarcinomas are positive. Two percent to 10% of mesotheliomas show focal staining. BG8 (Lewis Y ) Very useful. Approximately 90% to 100% of lung adenocarcinomas are positive. Three percent to 7% of mesotheliomas show focal reactivity. CEA (monoclonal) Very useful. Approximately 80% to 100% of lung adenocarcinomas are positive. Fewer than 5% of mesotheliomas are focally positive. B72.3 Very useful. Seventy-five percent to 85% of lung adenocarcinomas are positive. Very few mesotheliomas are positive. Ber-EP4 Very useful. Ninety-five percent to 100% of lung adenocarcinomas are strongly positive. Up to 20% of mesotheliomas are focally positive. TTF-1 Very useful. Seventy-five percent to 85% of lung adenocarcinomas show nuclear positivity. It is not expressed in mesotheliomas. Napsin A Very useful. Eighty percent to 90% of lung adenocarcinomas show cytoplasmic staining. It is not expressed in mesotheliomas. Abbreviations: BG8, blood group 8; CEA, carcinoembryonic antigen; TTF-1, thyroid transcription factor-1; WT-1, Wilms tumor 1. 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. Pleural Epithelioid Mesothelioma Versus Carcinoma 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 Table 6. Immunohistochemical Markers Used in the Differential Diagnosis Between Epithelioid Pleural Mesothelioma and Squamous Carcinoma of the Lung Marker Current Value/Comments Epithelioid mesothelioma (positive mesothelioma markers) WT-1 Very useful. Up to 95% of mesotheliomas show nuclear positivity. Lung squamous carcinomas are negative. Calretinin Somewhat useful. Virtually all mesotheliomas are positive, often strongly and diffusely, with nuclear and cytoplasmic staining. Approximately 40% of lung squamous carcinomas are positive, but the staining is often focal. D2-40 (podoplanin) Not useful. Approximately 80% to 100% of mesotheliomas are positive. Fifty percent of lung squamous carcinomas also stain. Cytokeratin 5 or 5/6 Not useful. It is expressed in 75% to 100% of mesotheliomas and 100% of lung squamous carcinomas. Lung squamous carcinoma (positive carcinoma markers) p63 or p40 Very useful. One hundred percent of lung squamous carcinomas show strong and diffuse nuclear positivity. Seven percent of mesotheliomas react, often focally. MOC-31 Very useful. Ninety-seven percent to 100% of lung squamous carcinomas are positive. Two percent to 10% of mesotheliomas show focal staining BG8 (Lewis Y ) Very useful. Eighty percent of lung squamous carcinomas are positive. Three percent to 7% of mesotheliomas show focal staining. Ber-EP4 Useful. Approximately 85% to 100% of lung squamous carcinomas are positive. Up to 20% of mesotheliomas are focally positive. Cytokeratin 5 or 5/6 Not useful. One hundred percent of lung squamous carcinomas and 75% to 100% of mesotheliomas are positive. Abbreviations: BG8, blood group 8; WT-1, Wilms tumor 1. Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al 657

12 Figure 11. A and B, Calretinin staining. A, Malignant mesothelioma has diffuse strong nuclear and cytoplasmic positivity. B, Adenocarcinoma is usually negative but may show focal positivity as shown here (original magnifications 3200 [A] and 3400 [B]). Figure 12. A and B, cytokeratin 5/6 staining. A, Malignant mesothelioma with strong reactivity. B, Large cell carcinoma with only focal reactivity (original magnifications 3400 [A and B]). 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 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, D Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al

13 Figure 13. A through C, Wilms tumor 1 (WT-1) staining. A, Strong nuclear staining in malignant mesothelioma invading fat. Note that endothelial cells show cytoplasmic staining only. B, Strong granular cytoplasmic staining in large cell carcinoma. C, Cytoplasmic staining in adenocarcinoma of lung (original magnifications 3200 [A] and 3400 [B and C]). (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. Immunohistochemical Issues in Peritoneal Mesothelioma 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 studies 68 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. Sarcomatoid Mesothelioma 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 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al 659

14 Figure 14. A and B, D2-40 staining. A, Strong membranous staining in malignant mesothelioma. B, Focal staining in squamous cell carcinoma (original magnifications 3200 [A] and 3400 [B]). Figure 15. A through C, MOC31 staining. A, Large cell carcinoma with membranous staining. B, Papillary adenocarcinoma of lung with strong staining. C, Focal staining in malignant mesothelioma (original magnifications 3400 [A and B] and 3200 [C]). Figure 16. Thyroid transcription factor-1 (TTF-1) shows strong nuclear staining in lung adenocarcinoma (original magnification 3400). 660 Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al

15 Figure 17. Blood group 8 (BG8) shows strong membranous staining in large cell carcinoma (original magnification 3400). Figure 18. PAX8 shows strong nuclear staining in this case of metastatic clear cell carcinoma from kidney (original magnification 3200). Figure 19. A, Fluorescence in situ hybridization (FISH) negative results for p16 deletion: 2 green signals (9p centromere) and 2 red signals (p16); B, FISH-positive results for p16 deletion: only 2 green signals (9p centromere) and no red signals (p16) (original magnifications [A and B]). 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 study 61 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 sar- Arch Pathol Lab Med Vol 137, May 2013 Malignant Mesothelioma Diagnosis Husain et al 661