Abstract
The adenomatoid tumor is an uncommon benign lesion, thus far described only in humans. Adenomatoid tumors typically arise in the genital tract, exceptionally in the heart, and usually represent an incidental finding. Microscopically, they are constituted by epithelioid cells that form tubular structures and anastomosing channels within a fibrous stroma. Mesothelial origin of these lesions is suggested by their immunohistochemical characteristics. In cattle, previously reported myocardial epithelial inclusions are morphologically similar in that the cells are immunoreactive for both cytokeratins and vimentin, and bear surface microvilli. Myocardial lesions found incidentally at slaughter in 8 cattle histologically resembled the so-called bovine myocardial epithelial inclusions and had morphologic and immunohistochemical features consistent with human adenomatoid tumor. All lesions were in the left ventricular myocardium, adjacent to the epicardium, and composed of epithelioid cells that formed cords and tubules, and were immunoreactive for pan-cytokeratins, cytokeratin 5/6, vimentin, calretinin, Wilms' tumor 1 suppressor gene, and CD30 antigen. By electron microscopy, numerous long slender microvilli were associated with desmosomes and tonofibrils. The immunohistochemical and ultrastructural features were considered consistent with mesothelial origin. These lesions, corresponding to the previously described myocardial epithelial inclusions in cattle, might be considered embryologic rests and could represent the bovine counterpart of the human adenomatoid tumor.
Introduction
Adenomatoid tumors (ATs) are considered benign mesothelial tumors that usually affect the human male 2,15,38 or female genital tract. 26,31 Rare extragenital ATs have been identified in the adrenal gland, 8,34 omentum, 13 pleura, 19 and lymph nodes. 16 A cardiac AT was described as an incidental finding in a man undergoing coronary artery bypass surgery. 25 The mesothelial origin of ATs has been established by immunohistochemical and ultrastructural studies. 5,17
The AT has never been described in animals, but similar lesions have been observed in the bovine heart and classified as myocardial epithelial inclusions (MEI). 1,12,18,27 Such cardiac lesions are incidental findings in slaughtered cattle and appear as single or multifocal masses of the left ventricle free wall or (rarely) of the interventricular septum. Histologically, MEI consist of acinar and tubular structures, embedded within dense collagen, and are immunoreactive for cytokeratins and, more or less, for vimentin. 1
The aim of this study was to investigate the morphologic features and the immunohistochemical differentiation in 8 cases of incidental lesions in bovine hearts. Light microscopic findings, immunoreactivity pattern, and ultrastructural features were consistent with a mesothelial differentiation and were similar to the AT as described in humans.
Materials and Methods
The signalment of the cattle is in Table 1. All 8 hearts were opened following the inflow and outflow tracts at the slaughter house during routine postmortem examination. Specimens of ventricular myocardium were sampled for microscopic examination, fixed in 10% buffered formalin for 24–48 hours, and processed for routine paraffin embedding. Sections of 3-µm thickness were stained with hematoxylin and eosin (HE). Additional sections were collected on poly-L-lysine-coated slides and processed for immunohistochemistry. Immunohistochemistry was performed using the standard avidin-biotin-peroxidase complex method, and specific antibodies against cytokeratin MNF116, cytokeratin 5/6, carcinoembryonic antigen (CEA), vimentin, calretinin, Wilms' tumor 1 suppressor gene (WT1), CD30, and factor VIII–related antigen (Table 2). For immunohistochemistry, sections were deparaffinized, treated with hydrogen peroxide, and heated in retrieval solution (citrate buffer solution, ph 6.0, 5 minutes × 3 passages in microwave oven at 650 W). Sections were then washed with phosphate-buffered saline (PBS), incubated for 60 minutes with the primary antibody, and rewashed with PBS. A biotin-labelled link antibody against mouse and rabbit immunoglobulins was applied for 30 minutes, followed by a streptavidin-biotin-peroxidase conjugate (LSAB, Dako). The chromogen was 3′-3′-diaminobenzidine-tetrahydrochloride. Sections were counterstained with Mayer's hematoxylin.
Signalment of cattle with myocardial adenomatoid tumor.
Immunohistochemical panel and immunostaining results.∗
∗HIER = heat-induced epitope retrieval; CEA = carcinoembryonic antigen; Factor VIII–RAg = factor VIII–related antigen.
Positive controls consisted of sections obtained from bovine lymph node, normal mesothelium (pericardium and peritoneum), thyroid tissue, segments of upper and lower gastrointestinal tract, kidney, adrenal gland, and abdominal ganglia. Negative controls were performed by replacing the respective primary antibodies by isotype and concentrations with matched irrelevant antibody.
For ultrastructural examination, representative fragments of myocardial lesions were retrieved from paraffin blocks and processed for electron microscopy. Briefly, fragments were put in xylene for 30 minutes with frequent changes to remove paraffin at room temperature, then in graded alcohol (absolute, 90%, 70%, and 50% ethanol, 20 minutes each), brought to water, and finally put in Sorensen's buffer for 20 minutes. The sections were postfixed in 1% osmium tetroxide, dehydrated in alcohol, and re-embedded in Araldite. Ultra-thin sections selected from the representative areas were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips 410 transmission electron microscope.
Results
Macroscopically, white, firm, plaquelike lesions were found in the free wall of the left ventricle in all cases, beneath the epicardial surface (Fig. 1). The lesions ranged from 0.5 to 1.5 cm in diameter. No other gross abnormality was detected at necropsy. Histologically, the lesions were unencapsulated and constituted by cords and tubules of large epithelioid cells with scant-to-moderate amounts of eosinophilic cytoplasm, uniform and round-to-oval nuclei, without atypia or significant mitotic activity (Fig. 2). Some tubular structures branched into anastomosing channels in abundant fibrous stroma with mild interstitial lymphoplasmacytic infiltration.
Bovine myocardium; animal No. 1. Cross-section of the left ventricular free wall. A white, firm, plaquelike lesion (1.0 cm × 1.5 cm) is adjacent to the epicardium. Bar = 0.5 cm.
Bovine myocardium, epithelioid lesion; animal No. 3. Cords and tubules of large epithelioid cells with scant-to-moderate amounts of eosinophilic cytoplasm, and uniform and round-to-oval nuclei. Some cords branch into anastomosing channels within abundant fibrous stroma. HE. Bar = 50 µm.
Immunohistochemical results are shown in Tables 2 and 3. In all lesions, the cells that formed cords and tubules had strong and diffuse cytoplasmic immunoreactivity for cytokeratin MNF116, vimentin, CD30 (in 4/8 cases, only focal in the remaining cases), and calretinin; multifocal and weak to strong cytoplasmic positivity for cytokeratin 5/6; and strong and diffuse nuclear immunoreactivity for WT1 antigen (Fig. 3a–d). None of the cells expressed either factor VIII–related antigen or CEA.
Immunoreactivity pattern of bovine myocardial adenomatoid tumors.∗
∗Immunoreactivity score: 0, negative (<5% positive cells), 1 (5-50% positive cells), 2 (>50-75% positive cells), 3 (>75% positive cells). CK = cytokeratin; FVIII–RAg = factor VIII–related antigen; CEA = carcinoembryonic antigen.
Bovine myocardium, epithelioid lesion; animal No. 4. Tubular and acinar cells have multifocal expression of cytokeratin 5/6 (a), and strong diffuse expression of calretinin (b), CD30 (c), and WT1 (d). Immunoperoxidase reaction, DAB chromogen and Harris' hematoxylin counterstain. Bar = 25 µm (a, b, c) and 30 µm (d).
Control bovine tissues had appropriate immunoreactivity. In particular, WT1 expression was observed in nuclei of normal mesothelium and renal glomeruli. Immunoreactivity for CD30 was observed in mesothelial cells and in a few scattered, mainly parafollicular, large lymphoid cells. Calretinin was expressed in mesothelium and in colonic neurons and nerves. Cytokeratin MNF116 and CEA immunoreactivity was found in colon epithelium, whereas cytokeratin 5/6 was expressed in nonkeratinizing squamous epithelium of the upper gastrointestinal tract. Vimentin and factor VIII–related antigen immunoreactivity was detected in various organs and tissues, properly localized to stromal mesenchymal cells and to endothelial cells, respectively. Immunoreactivity was abolished in negative controls for all antibodies.
Ultrastructurally, although a few artifacts from formalin fixation and paraffin embedding were present, the cells had a brush border of long slender microvilli (Fig. 4) and well-formed desmosomes associated with tonofibrils. On the basis of morphologic features and immunohistochemical results, these myocardial lesions were considered consistent with AT.
Bovine myocardium; animal No. 1. Transmission electron micrograph. Numerous long, branching microvilli (∗) are at the cell surface. Bar = 1.7 µm.
Discussion
The inclusions in each of 8 bovine hearts had the morphologic and immunohistochemical features of ATs in humans. These bovine cardiac lesions were incidental findings, as was the sole reported cardiac AT in a 53-year-old man undergoing coronary artery bypass surgery. 25 The unencapsulated white masses were in the left ventricular myocardium, adjacent to epicardium, and were constituted by epithelioid cells that formed cords, tubules, and anastomosing channels.
The immunoreactivity for cytokeratins, vimentin, CD30, calretinin, and WT1 antigen is consistent with a mesothelial differentiation. 36 This antibody panel has not been applied previously to bovine lesions; bovine pericardial and peritoneal mesotheliomas have been characterized immunohistochemically only by co-expression of cytokeratins and vimentin. 10,35 In the dog, mesotheliomas have been shown to be immunoreactive for pan-cytokeratin, cytokeratin 5/6, vimentin, calretinin, and WT1. 3,9,23,32,33 Ultrastructurally, elongated, thin microvilli at the cell surface, together with well-formed desmosomes and tonofibrils, support a mesothelial origin as in human ATs. 8,36
The AT is a rare benign lesion, first reported in 1942 21 and, again, in 1945. 11 Thus far, it has been reported in humans only, mainly in the genital tract, 2,15,26,31,38 with few reported extragenital cases and only 1 myocardial case. 25 A morphologically similar lesion of the bovine myocardium has been described as MEI. 1,12,18,27 We propose that the previously reported bovine MEI and the lesion that we have named AT might represent the same lesion. They share similar localization (myocardial, and mainly in the left ventricular free wall) and age distribution (from newborn to adult cattle), and both are generally considered incidental postmortem findings. 1,12,18,27
Bovine MEI have been hypothesized to be of endodermal origin on the basis of immunohistochemical (cytokeratin positivity in 8/8 cases, CEA in 3/8, vimentin in 3/8) and ultrastructural features. 1 We failed to detect CEA immunoreactivity in our series, and our results (co-expression of cytokeratin 5/6, vimentin, calretinin, WT1, and CD30), together with the ultrastructural features (microvilli, desmosomes, and tonofibrils), are consistent with mesothelial differentiation and mesodermal origin, as concluded in human ATs. 5,17,29 In the heart, ATs could originate from the epicardial surface by extension into the underlying myocardium. 25
In humans, WT1 protein 29 and calretinin 22,29 are considered mesothelial markers; they are expressed in mesotheliomas, but not in pulmonary adenocarcinomas. The WT1 gene encodes a zinc finger DNA-binding protein that acts as a transcriptional activator or repressor depending on the cellular or chromosomal context and that is required for the formation of the genitourinary system and mesothelial tissues. 37 The CD30 antigen, a 120 kD cytokine receptor that belongs to the tumor necrosis factor receptor (TNFR) superfamily, has been reported, not only in Reed-Sternberg cells of Hodgkin's disease, but also in other lymphoid and nonlymphoid cells and tissues and in related tumors (e.g., mesothelial, myoepithelial and decidual cells; lipoblasts; cultivated macrophages; mesotheliomas, soft tissue tumors, and histiocytic malignancies; and in reactive and neoplastic vascular lesions). 6 Cytokeratin 5/6 is usually expressed in epithelioid mesotheliomas, squamous cell carcinomas, and in many types of nonpulmonary carcinomas, whereas it is negative in pulmonary adenocarcinomas. 4 We did not detect CEA immunoreactivity in the bovine lesions. Lack of CEA reactivity is a criterion for identifying mesothelial proliferations, including mesothelioma, and the CEA reactivity that was reported in mesotheliomas in some early studies is nowadays attributed to a cross-reaction of the antibodies used in the past. 29 So far, CEA immunoreactivity has not been reported in ATs; 16 it has been reported in only 3/8 bovine MEI. 1
Cytokeratin 5/6 and calretinin immunohistochemistry has been validated in bovine claw epidermis, 14 pineal gland 28 and stellate ganglia. 24 In the present series, because immunohistochemistry was performed with commercial antibodies to human antigens, the antibodies were tested on positive and negative controls of bovine organs and tissues; all tests had appropriate results.
It is debatable whether myocardial AT is a true neoplasm or an embryologic rest. More importantly, these lesions are benign proliferations and should not be confused with malignant mesotheliomas or metastatic carcinomas. Malignant neoplasia can be excluded by the lack of significant cellular atypia. Endothelial origin is eliminated by the negativity of AT cells for endothelial markers. Another mesothelial lesion that has been described more often than the AT in the human heart, that is, the mesothelial/monocytic incidental cardiac excrescences (MICE), has distinctive clinical and histological features; these idiopathic benign exophytic proliferations are usually localized to valve leaflets and are constituted by epithelial cords and glandlike structures admixed with histiocytes, fibrin strands, neutrophils, and erythrocytes. 30 Another entity that has been described in humans, the congenital polycystic tumor (also called endodermal heterotopia or mesothelioma), is localized to the atrioventricular (AV) node. 7,20 It usually presents with an AV block or as sudden death and comprises polycystic structures lined by cuboidal, transitional, and/or squamous cells that are usually immunoreactive for cytokeratins and CEA, and negative for calretinin. 39 This immunoreactivity makes mesothelial origin unlikely, and the polycystic tumor of the AV node is nowadays considered endodermal foregut tissue that has been displaced during embryogenesis. 7,20,39
In conclusion, histologic features of the myocardial lesions in these 8 cattle are similar to those described for bovine myocardial epithelial inclusions, which were thought to be derived from endoderm. However, immunohistochemical and ultrastructural features resemble those of human adenomatoid tumors and are consistent with mesothelial differentiation and mesodermal origin.