Immunohistochemistry Case Examples
Allen M. Gown
Anthony S.Y. Leong
Section 2 -
Peritoneal mesothelioma, foamy cell variant
Anthony S.Y. Leong
University of Newcastle
A 69-year-old man first presented for general malaise and was found to have a large spleen.
Peripheral blood counts were within normal limits and a 1265 g, 205 x 150 x 85mm spleen was removed. A
3mm thick layer of clearly demarcated tumor encased the spleen. The tumor was composed of sheets of
large polygonal cells with abundant eosinophilic granular to clear cytoplasm. Nuclei were vesicular and
contained prominent eosinophilic nucleoli. Mitotic activity was low (Figs 1, 2 and 3). Focal
collections of pigment containing cells were present (Fig 4). Large foam cells were frequently present
in the red pulp of the spleen (Fig 5). Immunostaining showed positivity for broad-spectrum cytokeratin
with Cam 5.2 and the tumor was negative for S100. The diagnosis rendered was "metastatic carcinoma,
clear cell type, a renal primary cannot be excluded".
The patient represented in 44 months later with subacute intestinal obstruction and laparotomy
revealed the greater omentum to be caked by a glistening opaque grey tumor with smaller deposits
scattered in the peritoneum. An omental biopsy of 145 x 60 x 25 mm was obtained.
The tumor cells were large and pleomorphic and showed abundant, foamy and eosinophilic granular
cytoplasm with distinct cytoplasmic borders. They had vesicular nuclei with prominent eosinophilic
nucleoli and occasional multinucleated cells were present. The tumor cells formed sheets, clusters and
cords and focally micropapillary projections and gland-like structures were present.
Immunostaining revealed positivity for calretinin, WT1, and CK5/6. EMA revealed distinct long
microvilli on the surface of the tumor cells, often circumferential and aberrant in location, juxtaposed
to collagen and stroma. Intra-cytoplasmic lumina were also displayed. D2-40 was positive, similarly
highlighting surface microvilli. HBME1 stain was similarly positive. PAS/D, CEA, S100 and HMB45 were
negative. The splenic foam cells were CD68 positive and did not bear any mesothelioma markers.
Diagnosis: Peritoneal mesothelioma, foamy cell variant.
The diagnosis of mesothelioma, especially with unusual clinical presentation or histology, continues
to be a challenge. Four main histologic subtypes of diffuse malignant mesothelioma are recognised.
These include epithelial (including well-differentiated papillary), sarcomatous (including desmoplastic),
biphasic and poorly differentiated, the majority (75%) of peritoneal mesotheliomas being of the
epithelial subtype. In addition, unusual variants have been described and include lympho-histiocytoid,
small cell, deciduoid, adenoid cystic, and a clear cell and signet ring (lipid-rich) pattern, with the
localised mesothelioma and mesothelioma of tunica vaginalis being included as variant clinical
Recently, a 56-year-old woman with no history of asbestos exposure was reported with a pleural
mesothelioma of solid and tubulo-papillary pattern in which the tumor cells were large with vesicular
nuclei and prominent nucleoli.  Cytoplasm was abundant and mostly eosinophilic but in about
40% of the neoplastic cells it was clear and foamy. Mitotic activity was low. The foamy nature of the
tumor cells was shown to be due to collections of lipid droplets by electron microscopy. Foamy cell
mesothelioma is very uncommon and should not be mistaken for its mimics. Foamy cell change has been
recognised in carcinomas and foam cells may often be macrophages. Foamy macrophage-like neoplastic
mesothelioma cells have been described in pleural effusions in three mesotheliomas in which subsequent
histologic specimens revealed mesotheliomas of conventional histology with no foamy elements. 
Rare foamy cells were described in a papillary mesothelioma of the tunica vaginalis  and clear
cell change has been reported in mesotheliomas.
Foamy macrophages have also been reported
as a feature of a peritoneal mesothelioma which did not show foamy cell change.  More
recently, an ultrastructural study of mesothelioma with clear cell features revealed that the most
frequent cause of cytoplasmic clearing was the accumulation of large amounts of glycogen. Accumulation
of cytoplasmic lipid alone or in conjunction with glycogen was a less common factor and other causes
included marked mitochondrial swelling, the presence of intracytoplasmic vesicles and large numbers of
intracytoplasmic lumens with microvilli. 
The present case showed striking foamy and granular cells as a major component of the mesothelioma.
This contributed to the failure to recognise the tumor initially and there were also striking foamy
macrophages in the spleen.
The differential diagnosis of malignant mesothelioma includes metastatic carcinoma and reactive
mesothelium. Despite the increasing number of markers to separate these entities, some authors still
feel that "immunohistochemical analysis still has a limited role in the diagnosis of malignant
mesothelioma".  Calretinin, thrombomodulin, keratin 5/6, mesothelin and WT1 are more recent
additions to the list of mesothelioma markers but each has varying sensitivity and specificity, none
being 100% specific, with lowest sensitivity in sarcomatoid mesotheliomas.  As such, a panel
of antibodies is essential for the diagnosis of mesothelioma (Table 1).
Table 1: Antibody panel for mesothelioma, adenocarcinoma and reactive
| ||vim ||CK5/6 ||Calret ||BerEP4 ||CEA ||Throm ||WT1 ||mesoth|
|Mesothelioma ||+ ||+ ||+ ||-/+ ||- ||+ ||+ ||+|
|Adenocarcinoma ||-/+ ||+/- ||-/+ ||+/- ||-/+ ||+/- ||-/+||+/-|
|Reactive mesoth ||+/- ||+/- ||-/+ ||- ||- ||+/- ||+ ||+/-|
Calretinin is expressed in the majority of epithelial mesothelioma but is also common in small and
large cell carcinomas of the lung.  Thrombomodulin and CK5/6 are also expressed in the great
majority of keratinising and non-keratinising carcinomas of the lung.  TTF-1 is expressed
exclusively by pulmonary carcinomas especially adenocarcinoma and thyroid tumors and should be included
in the panel. Importantly, calretinin and mesothelin may be positive in about 1/3 of thymic
carcinomas  (and thymomas) which can also present in the pleural cavity.
The diagnostic ultrastructural features that identify malignant mesothelioma from adenocarcinoma and
reactive mesothelium include long microvilli that are devoid of core rootlets, aberrant long microvilli
that are juxtaposed to stromal collagen, and microvilli coated with proteoglycan particles. Optimal
staining for EMA can highlight such microvillous processes,  initially described as "thick
membrane" staining.  Circumferentially disposed microvilli can also be demonstrated with EMA,
correlating with the aberrant microvilli seen at ultrastructural level, a feature that is pathognomonic
of malignant mesothelial cells.  Such long microvilli can also be demonstrated in
intercellular and intracellular lumina with anti-EMA. In our hands, this marker is extremely useful for
the identification of malignant mesothelioma, especially of the epithelial subtype. The more recent use
of HMBE1 serves a similar purpose of highlighting the long microvillous processes. 
and podoplanin  are two most recently developed markers for lymphatic
endothelial cells that have been shown to mark mesothelial cells and mesotheliomas. The antigens are
localised to the cell membrane and also produce a pattern of staining which highlights the long surface
microvilli of mesotheliomas.
- Allen TC. Recognition of histopathologic patterns of diffuse malignant mesothelioma in differential diagnosis of pleural biopsies. Arch Pathol Lab Med 2005;129:1415-20.
- Cavazza A, Pasquinelli G, Agostini I, et al. Foamy cell mesothelioma. Histopathology 2002;41:369-71.
- Spriggs AL, Grunze H. An unusual cytologic presentation of mesothelioma in serous effusions. Acta Cytol 1983;27:288-92.
- Mikuz G, Hopfel-Kreiner I. Papillary mesothelioma of the tunica vaginalis propria testis. Case report and ultrastructural study. Virchows Arch A Pathol Anat Histol 1982;396:231-8.
- Ordonez NG, Mackay B. Glycogen-rich mesothelioma. Ultrastruct Pathol 1999;24:401-6.
- Dessy E, Falleni M, Braidotti P, et al. Unusual clear cell variant of epithelioid mesothelioma. Arch Pathol Lab Med 2001;125:1588-90.
- Kitazawa M, Kab]neko H, Toshima M, et al. Malignant peritoneal mesothelioma with massive foamy cells. Cod fish roe-like mesothelioma. Acta Pathol Jpn 1984;34:687-92.
- Ordonez NG. Mesothelioma with clear cell features: an ultrastructural and immunohistochemical study of 20 cases. Hum Pathol 2005;36:465-73.
- Roberts F, Harper CM, Downie I, Burnett RA. Immunohistochemical analysis still has a limited role in the diagnosis of malignant mesothelioma. A study of thirteen antibodies. Am J Clin Pathol 2001;116:253-62.
- Leong AS-Y, Leong FJW-M, Cooper K. Manual of Diagnostic Antibodies for Immunohistology. 2nd. London: Greenwich Medical Media, 2003.
- Miettinen M, Sarlomao-Rikala M. Expression of calretinin, thrombomodulin, keratin 5, and mesothelin in lung carcinomas of different types; an immunohistochemical analysis of 596 tumors in comparison with epithelioid mesotheliomas of the pleura. Am J Surg Pathol 2003;27:150-8.
- Pan CC, Chen PC, Chou TY, Chiang H. Expression of calretinin and other mesothelioma-related markers in thymic carcinoma and thymoma. Hum Pathol 2003;34:1155-62.
- Leong AS-Y, Vermin-Roberts E The immunohistochemistry of malignant mesothelioma. Pathology Annual 1994;29(II): 157-179.
- Leong AS-Y, Parkinson R, Milios J. "Thick" cell membranes revealed by Immunocytochemical staining: A clue to the diagnosis of malignant mesothelioma. Diagn Cytopath 1990;6:9-13.
- Leong AS-Y, Wick ME, Swanson P. Immunohistology and Electron Microscopy of Anaplastic and Pleomorphic Tumors. Cambridge: Cambridge University Press, 1997.
- Dahlstrom JE, Maxwell LE, Brodie N, et al. Distinctive microvillous brush border staining with HBME-1 distinguishes pleural mesotheliomas from pulmonary adenocarcinomas. Pathology 2001;33:287-91.
- Odonez NG. D2-40 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma. Hum Pathol 2005;36:372-80.