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In 1978, Beckwith and Palmer1 identified a group of renal tumors occurring in infants and young children that were characterized by a proliferation of noncohesive or loosely cohesive cells having abundant cytoplasm, a large eccentric nucleus with a prominent nucleolus, and a hyaline intracytoplasmic inclusion displacing the nucleus. Since subsequent ultrastructural2, 3 and immunohistochemical4 investigations of these cells did not show any evidence of rhabdomyoblastic differentiation, the descriptive term rhabdoid was introduced to designate these neoplasms,2 thus emphasizing their resemblance to rhabdomyosarcomas. Following these publications, numerous reports of tumors having rhabdoid cells and occurring in a wide variety of extrarenal locations appeared in the literature which resulted in considerable debate regarding the nomenclature and histogenesis of these tumors. Current information indicates that rhabdoid tumors exist both as a specific entity and as a secondary morphologic phenotype that is encountered within a wide array of tumor types originating in a variety of locations, typically indicating the development of cytologic anaplasia and aggressive biological behavior. The tumors in the latter group characteristically show a mixture of a recognizable parent neoplasm admixed with a rhabdoid component and are usually referred to as composite rhabdoid tumors.5

A well-known characteristic of mesotheliomas is their ability to exhibit a broad range of cytomorphologic features and to grow in a wide variety of histologic patterns. Based on their light microscopic appearance, these tumors have been subdivided into epithelioid, sarcomatoid, mixed epithelioid and sarcomatoid (biphasic), and desmoplastic types. Although epithelioid mesotheliomas more often present a tubulopapillary or solid pattern, on occasion, they may present other patterns, including deciduoid, clear cell, signet ring, and pleomorphic. Mesotheliomas presenting rhabdoid features are rare and, to my knowledge, only two individual case reports of such tumors have been published.6, 7 Since mesotheliomas with rhabdoid features can potentially be confused with a variety of other tumors with similar morphologic features, especially in small biopsies, the purpose of this study is to make pathologists aware of the existence of this uncommon morphologic variant of mesothelioma and also to discuss the value of electron microscopy and immunohistochemistry in its diagnosis.

Materials and methods

A total of 10 mesothelioma cases were obtained from the files of the Department of Pathology, and the Electron Microscopy section at the University of Texas MD Anderson Cancer Center, and from the personal consultation files of the author. The specimens consisted of three pleural biopsies, one decortication specimen, six extrapleural pneumonectomy specimens, and biopsies from the omentum and periumbilical mass in one case, and autopsy material in one case.

Tissue specimens were fixed in 10% buffered formalin and processed for routine light microscopy. Tissue sections from the paraffin blocks were stained with hematoxylin and eosin. Immunohistochemical studies were carried out on formalin-fixed, paraffin-embedded tissue sections using the avidin–biotin–peroxidase complex method in a Dako AutoStainer (Carpinteria, CA, USA). The primary antibodies that were used are listed in Table 1. The immunostaining was carried out using the LSAB2 peroxidase kit (Dako). To enhance the immunostaining, a heat epitope retrieval procedure was performed using a Black-and-Decker (Shelton, CT, USA) vegetable steamer. Briefly, deparaffinized sections were placed in a thermoresistant container filled with a buffer solution, steamed for 45 min, then cooled for 20 min before immunostaining. Depending on the antibody, the buffer solutions used were either sodium citrate buffer, pH 6.0, or a 10:1 solution of Tris-EDTA buffer, pH 8.0. Enzymatic pretreatment with 0.2% protease, type XXIV, (Sigma Chemical, St Louis, MO, USA) in Tris-EDTA buffer saline, pH 7.3, at room temperature for 2 min, was used with the Ber-EP4 antibody. The antigen–antibody reaction was visualized using 3-amino-9-ethylcarbazole or 3,3′-diaminobenzidine tetrahydrochloride as chromogen. To evaluate the specificity of the antibodies, known positive and negative tissues were used as controls. The immunostaining was graded on a sliding scale of 1+ to 4+ according to the percentage of reactive cells (trace, <1%; 1+, 1–25%; 2+, 26–50%; 3+, 51–75%; 4+, 76–100%). Electron microscopy studies were performed in nine of the cases. Samples of the specimens were fixed in 2% buffered glutaraldehyde and embedded in Epon epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate.

Table 1 Antibodies used in this study
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Results

Clinical Findings

Eight patients were men and two were women ranging in age from 52 to 79 years (mean, 62.8 years). There was a history of asbestos exposure in six patients and smoking in three. The mesothelioma originated in the pleura in nine cases and in the peritoneum in one. Two patients received chemotherapy alone; five underwent extrapleural pneumonectomy with four of these also receiving radiation therapy and one chemotherapy. One patient underwent decortication. Treatment information was not available in the remaining two patients. All six patients for whom follow-up information was available died of disease 3–12 months after diagnosis (mean, 5.7 months).

Pathology Findings

Gross examination of the five pneumonectomy specimens showed diffuse involvement of the visceral and parietal pleura with encasement of the lung. In two of the cases (cases 7 and 8), several of the peribronchial lymph nodes were involved by metastases. The most significant light microscopic findings are summarized in Table 2. Histologically, seven of the mesotheliomas were epithelioid, two sarcomatoid, and one biphasic. The proportion of the rhabdoid component in these tumors ranged from 15 to 75%, and all were 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 caused nuclear indentation (Figure 1).

Table 2 Summary of clinical and pathology findings
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Figure 1
figure 1

Case 8: (a) Rhabdoid cells arranged in an alveolar pattern. (b) Higher magnification showing discohesive cells with rhabdoid features. Case 7: (c) In this mesothelioma, the rhabdoid cells are primarily located in the areas of the tumor with myxoid stroma. (d) Higher magnification showing rhabdoid cells with typical cytoplasmic inclusions. (e) Case 10: Sarcomatoid mesothelioma showing an area of discohesive cells exhibiting rhabdoid morphology. (f) Higher magnification of the rhabdoid cells.

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Immunohistochemistry

The immunohistochemical results are summarized in Table 3. The rhabdoid cells in all 10 cases strongly reacted for vimentin and pan-keratin, and a globoid-like inclusion was often apparent in the neoplastic cells (Figures 2a and b). Expression for keratin 7 was seen in all eight of the cases stained for this marker and in most instances, the reactivity was strong and diffuse (Figure 2c). The rhabdoid cells also stained for calretinin in nine of 10 cases and for keratin 5/6 in seven of nine (Figure 2d). These cells also exhibited nuclear positivity for WT1 in four of seven cases, and membranous reactivity for mesothelin in four of six and podoplanin in two of six (Figures 2e and f). Only one case showed focal positivity for desmin, but the staining occurred in the nonrhabdoid cells. None of the cases stained for muscle-specific actin, bcl-2, CD15, CD34, TAG-72 (B72.3), CEA, MOC-31, or TTF-1 were positive for any of these markers in either the rhabdoid cells or in the nonrhabdoid component of the tumors.

Table 3 Immunohistochemical results
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Figure 2
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(a) Case 10 showing strong reactivity for vimentin in the rhabdoid cells. (b) The same case as shown in (a) showing positivity for pan-keratin in both the rhabdoid and spindle cells. (c) Case 9 exhibiting strong keratin 7 expression in the rhabdoid cells. (d) Case 6 showing nuclear and cytoplasmic positivity for calretinin in the rhabdoid cells. (e) The same case as shown in (d) showing membranous reactivity for mesothelin in some of the rhabdoid cells. (f) Case 3 showing membranous positivity for podoplanin in the rhabdoid cells.

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Electron Microscopy

Electron microscopy demonstrated that the rhabdoid cells had a peripherally placed nucleus often containing a prominent nucleolus (Figures 3a, b, and 4). The cytoplasm was abundant and contained paranuclear collections of intermediate filaments which occupied most of the cytoplasm. These intermediate filaments were arranged in either interlacing bundles or a whorl-like array (Figures 3b and 4). In some instances, a variable number of organelles appeared entrapped within the intermediate filaments (Figure 3b). The cell membrane of the cells showing early rhabdoid changes were covered by a variable number of short microvilli which were usually seen along the apical and lateral surfaces of the cells (Figures 3a and b).

Figure 3
figure 3

(a) Electron micrograph showing two tumor cells with early rhabdoid features. In both cells, the nuclei are eccentrically placed and the one on the left contains a large nucleolus. A vague globular-like collection of intermediate filaments in the cytoplasm of the cell on the right is also apparent. Only a few short microvilli are seen on the cell membrane of both cells. (b) Tumor cell showing a globoid aggregate of paranuclear cytoplasmic filaments producing compression of the nucleus. A few mitochondria and short profiles of endoplasmic reticulum are seen within the collection of the intermediate filaments. The intercellular space appears dilated and the cell membrane is covered by microvilli. (a) × 6700; (b) × 10 000

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Figure 4
figure 4

High magnification of a rhabdoid cell showing a large collection of intermediate filaments corresponding to the paranuclear inclusions seen in the rhabdoid cells on light microscopy. The cell membrane appears completely devoid of microvilli (× 12 000).

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Discussion

Although malignant rhabdoid tumors were first described in the kidney and were initially considered to represent an especially aggressive Wilms’ tumor with a rhabdomyosarcomatous pattern, examples of tumors exhibiting rhabdoid features were subsequently reported in a wide variety of other anatomic sites including the central nervous system,8, 9 skin,10, 11 liver,12, 13 gastrointestinal tract,14 lung,15, 16, 17, 18 bladder,19, 20 thymus,21 tongue,22 vulva,23 and soft tissue,24, 25, 26 and as a variant of melanoma.27, 28, 29 At present, only malignant rhabdoid tumors of the kidney and atypical teratoid/rhabdoid tumors of the central nervous system are well-defined entities.30, 31, 32 These tumors are characterized by having a predilection for infants and young children, aggressive clinical behavior with short survival time, a polyphenotypic immunoprofile, and characteristic deletions and mutations involving the INI1/hSNF5 tumor suppressor gene on chromosome 22q11.2.33, 34 It has been suggested, however, that some other subgroups may exist, especially among those malignant rhabdoid tumors originating in the liver and soft tissue, because they share specific gene alterations and may, therefore, represent specific entities.35, 36 It should be emphasized, though, that current evidence indicates that the large majority of extrarenal malignant rhabdoid tumors, excluding those previously mentioned, represent a distinct phenotype that is shared by a number of tumors as they undergo anaplastic progression.

Mesotheliomas exhibiting rhabdoid features are rare and only two such cases have been documented in the literature.6, 7 The first case, which was reported by Matsukuma et al in 1996,6 was a localized peritoneal malignant mesothelioma in a 68-year-old man with no history of asbestos exposure. Histologically, the tumor was a biphasic mesothelioma containing sarcomatoid cells with rhabdoid features. Immunohistochemical studies demonstrated vimentin, keratin, and epithelial membrane antigen expression in the rhabdoid cells. No reactivity for desmin, myoglobin, actin, or S-100 protein was observed. Electron microscopy studies showed globular aggregates of intermediate filaments but there was no evidence of rhabdomyoblastic differentiation. The second case was a pleural mesothelioma that was reported by Puttagunta et al in 2000.7 The patient was a 41-year-old man with no occupational history of asbestos exposure who had received radiotherapy for mediastinal Hodgkin's disease 14 years earlier. The histological appearance of this tumor was that of a deciduoid mesothelioma with focal rhabdoid change. Immunohistochemical studies showed positive staining for keratin, vimentin, and calretinin, and negative staining for CEA, Ber-EP4, and CD15 (leu-M1). Ultrastructural studies demonstrated paranuclear aggregates of intermediate filaments arranged in a concentric pattern.

The results of the present investigation demonstrated that, in general, the rhabdoid cells tended to maintain the immunophenotype seen in the nonrhabdoid areas of the tumor. In all of the cases, these cells retained the strong expression for vimentin, pan-keratin, keratin 7, and calretinin that was also seen in the areas of the tumors exhibiting a more conventional morphology. Other mesothelioma markers, specifically keratin 5/6, WT1, mesothelin, and podoplanin, were also demonstrated, but their expression was less frequent and when it occurred, the percentage of cells in the rhabdoid areas expressing these markers was lower when compared with the nonrhabdoid areas of the tumor. These findings correlate with the ultrastructural demonstration of varying degrees of mesothelial differentiation in the rhabdoid cells. While the more differentiated cells had microvilli on the cell membrane and relatively small nodular aggregates of intermediate filaments, the cell membrane of the less differentiated cells was devoid of microvilli and most of the cytoplasm was occupied by a large collection of intermediate filaments arranged in interlacing fascicles or in a concentric array.

One of the immunohistochemical markers that was found to be expressed in mesotheliomas with rhabdoid features and which merits additional comment is the novel mesothelioma marker podoplanin. Although podoplanin and the recently commercially available D2-40 monoclonal antibody that was raised against an unidentified M2A protein derived from germ cell tumors were initially considered to be two different mesothelioma markers, recent investigations have shown that D2-40 specifically recognizes podoplanin,37 a 38 kDa mucin-type transmembrane protein that was first detected on the surface of the rat glomerular epithelial cells (podocytes) and was found to be linked to the flattening of the foot processes in puromycin-induced nephrosis.38 As podoplanin was found to be expressed in 86–100% of the epithelioid mesotheliomas, but absent in lung adenocarcinomas, recent investigations have indicated that immunostaining for this protein could be very useful in distinguishing between these two malignancies.39, 40, 41, 42 It should be mentioned, however, that in addition to epithelioid mesotheliomas, podoplanin expression has been reported in a subset of angiosarcomas,43, 44 in a minority of serous carcinomas,39, 45 in squamous carcinomas of the lung,42, 46 and in the epithelioid component of biphasic synovial sarcomas.41 All of these tumors can potentially be confused with mesotheliomas.

It is important for pathologists to be aware that mesotheliomas can exhibit rhabdoid features and can, therefore, potentially be confused with a variety of tumors with similar morphology that can involve the serosal membranes. The four tumors with the greatest potential of being confused with mesotheliomas with rhabdoid features are lung carcinomas with rhabdoid features, proximal-type epithelioid sarcomas, synovial sarcomas, and rhabdomyosarcomas. Carcinomas of the lung can present rhabdoid features and can involve the pleura. In over half of the published cases of these tumors in which the status of the pleura was mentioned, this structure was reported as being involved by tumor.15, 16, 17, 47 Rare examples of lung carcinomas with rhabdoid features diffusely involving the pleura and clinically and radiologically mimicking mesothelioma have also been documented in the literature.15 Immunohistochemical studies can assist in establishing the differential diagnosis. As was demonstrated in this study, mesotheliomas with rhabdoid features often express mesothelial markers (ie, calretinin, WT1, podoplanin, mesothelin) that are usually absent in carcinomas.41, 48 It should be mentioned, however, that TTF-1, a marker that is frequently expressed in lung carcinomas,48, 49 is usually absent in carcinomas of the lung with rhabdoid features.18 In a recent investigation, no reactivity for TTF-1 was seen in the rhabdoid areas of 11 carcinomas of the lung with rhabdoid features and only three of these 11 cases focally expressed this marker in the nonrhabdoid component of the tumor.18 Additionally, while keratin 7, which is usually present in both mesotheliomas and adenocarcinomas of the lung, was found to be strongly expressed in the rhabdoid component of the mesotheliomas, it was rarely demonstrated in the rhabdoid component of the lung carcinomas.

Mesotheliomas with rhabdoid features can also be confused with proximal-type epithelioid sarcoma, a tumor that is primarily composed of rhabdoid cells and which, like mesotheliomas, can originate in the inguinal region.50, 51 Similar to mesotheliomas, the rhabdoid cells in epithelioid sarcomas express vimentin and keratin and on occasion, some mesothelioma markers such as keratin 5/6 and calretinin.52 However, in contrast to mesotheliomas, epithelioid sarcomas frequently express CD34,52, 53, 54 a marker that has been reported to be invariably absent in mesotheliomas.55, 56

Synovial sarcomas can originate in or metastasize to the pleura or the chest wall and, like mesotheliomas, may present a wide variety of morphologic appearances, including a rhabdoid morphology.57, 58 Additionally, these tumors may also express some mesothelioma markers, including calretinin, mesothelin, and podoplanin.41, 59 However, synovial sarcomas do not exhibit positivity for WT1, a marker that is often expressed in mesotheliomas; therefore, immunostaining for the WT1 protein may assist in establishing the differential diagnosis between these malignancies.59 Another immunohistochemical marker that can also be useful in this differential diagnosis is bcl-2 which has been reported to be expressed in 79–100% of the synovial sarcomas, but in only 0–10% of the mesotheliomas.60, 61, 62, 63 The diagnosis of synovial sarcoma can also be established by the demonstration of the distinctive t(x;18)(p11;q11) translocation that has been reported to be present in nearly all of the cases.64, 65

Finally, mesotheliomas with rhabdoid features can be distinguished from rhabdomyosarcomas by the expression of mesothelial markers in the mesotheliomas or by the demonstration of rhabdomyoblastic markers, such as myogenin or MyoD1, in rhabdomyosarcomas. These rhabdomyoblastic regulatory proteins are often expressed in rhabdomyosarcomas,66, 67 but are absent in mesotheliomas.68 Desmin is another marker that is frequently used in the diagnosis of rhabdomyosarcomas and, although it has been reported to be present in mesotheliomas, controversy exists regarding the percentage of desmin positivity in the latter tumors. While some groups of investigators have reported expression of this protein in 41–88% of the epithelioid69, 70, 71 and in 33–71% of the sarcomatoid69, 70, 72 mesotheliomas, others have reported desmin positivity in 0–10% of the epithelioid68, 73, 74, 75, 76 and in none of the sarcomatoid.74, 75, 76 Of the eight cases investigated for desmin expression in the present study, only one exhibited positivity in sparse groups of neoplastic cells in the nonrhabdoid component of the tumor, a finding which confirms previous observations indicating that desmin can be expressed in mesotheliomas but it is a relatively uncommon phenomenon. Electron microscopy can also be useful since rhabdomyosarcomas would exhibit rhabdomyoblastic differentiation.

The results of the present investigation suggest that the presence of rhabdoid features in a mesothelioma is an indication of particularly aggressive behavior as shown by the short survival time of the six patients for whom follow-up information was available. With the exception of one patient who survived for 1 year after diagnosis, all of the remaining patients died within a 6-month period (mean survival, 3.8 months). A further indication of the aggressive behavior of these tumors was the extensive lymph node involvement that was found in the pathology specimens of two of the five patients who underwent extrapleural pneumonectomy. Perhaps this aggressive behavior should not be surprising, however, given that other tumors originating in a variety of locations and exhibiting the rhabdoid phenotype have often been reported as being unusually aggressive when compared with their more histologically conventional counterparts. In order to fully determine the prognostic significance of the rhabdoid phenotype in mesotheliomas, more studies with larger numbers of cases are needed.