Ultrastructural Pathology, 36(1), 23–30, 2012 Copyright © 2012 Informa Healthcare USA, Inc. ISSN: 0191-3123 print/1521-0758 online DOI: 10.3109/01913123.2011.631725

original article

Mesenchymal–Epithelial Differentiation of Adamantinoma of Long Bones: An Immunohistochemical and Ultrastructural Study Carmen D. Sarita-Reyes, MD1, M. Alba Greco, MD2, and German C. Steiner, MD3

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1 Department of Pathology and Laboratory Medicine, Boston Medical Center, Boston, MA, USA, 2Department of Pathology, NYU Langone Medical Center, New York, New York, USA, and 3Department of Pathology and Laboratory Medicine, NYU Hospital for Joint Diseases, Langone Medical Center, New York, New York, USA

Ab stract Three cases of adamantinoma were studied by electron microscopy and immunohistochemistry. In the tubular pattern, well-differentiated epithelial cells and glandular structures were present, in addition to ill-defined glands. In the basaloid pattern, less differentiated epithelial cells with discohesion were seen in the central epithelial masses. This study established the epithelial nature of some tubular structures with slit-like lumina, easily misinterpreted as capillaries by light microscopy. Results also showed that the irregular spaces observed within the basaloid pattern probably result from cell discohesion. Moreover, this investigation demonstrates the epithelial nature of a subset of spindle cells within the stroma of adamantinoma and offers ultrastructural evidence for a probable mesenchymal–epithelial transformation as its histogenesis. Keywords:  Adamantinoma, electron microscopy, mesenchymal–epithelial differentiation

Introduction

We performed electron microscopy studies of three cases of classic adamantinoma with the following purposes: first, to describe in greater detail the tubular pattern and the central epithelial masses with gland-like spaces of the basaloid pattern; second, to study the stromal cell component of the tumor; and third, to determine the presence of early epithelial differentiation in adamantinoma and its possible origin from the stromal cells.

Adamantinoma of long bones is a rare low-grade malignant epithelial neoplasm that more frequently affects the tibia [1]. Histologic patterns include basaloid, tubular, spindle cell, squamous, and osteofibrous dysplasia-like variants [2]. Early articles published on electron microscopy (EM) studies [3–7] have conclusively established the epithelial nature of this tumor. Immunohistochemistry (IH) studies of adamantinoma also have confirmed its epithelial nature [8,9]. Most EM articles published on adamantinoma have described a basaloid pattern [10–16], but there is scant illustration of the tubular or glandular pattern [17]. To our knowledge, none of the EM reports of adamantinoma reviewed in the literature were published in the journal Ultrastructural Pathology. The pathogenesis of adamantinoma is not known, and it is not clear how the epithelial cells originate from within the bone. In a recent immunohistochemical study of the extracellular matrix of adamantinoma, it has been suggested that the epithelial component may originate from the fibrous component of the tumor by a mesenchymal–epithelial transformation [18].

Materials and Methods Three cases of adamantinomas of the tibia were retrieved from the archive material of the Deparment of Pathology, NYU Hospital for Joint Diseases.

Case Histories Case 1 A 55-year-old male presented in 1991 with low back pain for few months and also some pain in the right tibia. A bone scan showed increased activity in the

Received 12 August 2011; revised 03 October 2011; accepted 06 October 2011 Correspondence: German C. Steiner, MD, Department of Pathology, NYU Hospital for Joint Diseases, 301 17th Street, New York, NY 10003, USA. E-mail: [email protected]

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24    C. D. Sarita-Reyes et al.

Figure 1  Lateral radiograph of the tibia showing an anterior intracortical tumor with bone expansion, lytic and sclerotic areas.

right tibia, and radiographs demonstrated a lesion in the anterior tibial cortex (Figure 1). The patient had a biopsy, which was followed by segmental resection of the tibia with allograft reconstruction. Cut sections of the specimen demonstrated an anterior intracortical tumor measuring 6 × 2 cm. containing yellowish soft tissue admixed with sclerotic bone. On follow-up 18 years later, the patient was well and free of tumor with no pain and complete remodeling of the allograft. Case 2 A 25-year-old female presented in 1989 with a 1-year history of a bump on her left leg, with recent enlargement and pain. She denied any previous injury, surgery, or any other medical problem. On physical examination she had anterior enlargement of the tibia with no soft tissue mass. Radiographs revealed a tumor within the tibia (Figure 2). The patient underwent curettage, which was followed by resection of the midshaft of the tibia and allograft reconstruction. Cut sections of the specimen showed cortical bulging and an intramedullary yellowish tumor measuring 5.5 cm in length with cortical thinning, without soft tissue extension. No clinical follow-up is available on this patient. Case 3 A 28-year-old female patient, who was initially seen in another country in 1980, presented with an 8.5 × 2.5-cm

Figure 2  Lateral view of the tibia demonstrating a lytic and expansile tumor involving the anterior cortex and medullarity cavity with cortical thinning. Focal sclerosis is present within the lesion.

tumor involving the right tibial diaphysis. A bone biopsy was performed, with a pathologic diagnosis of fibroxanthoma, questionable nonossifying fibroma. Curettage of the medullary cavity was performed with bone grafting. Four years after the initial surgery in 1984, the patient noticed swelling and pain in the leg (Figure 3). A biopsy was performed, and she was referred to our institution for further treatment. Only the biopsy tissue from 1984 was available for review and was diagnosed as a classic adamantinoma. The patient then had surgical resection of the entire tibia and reconstruction with the fibula and tibial allograft. Cut sections of the specimen revealed an intramedullary tumor measuring 22 cm in length, which occupied the diaphysis of the tibia. A large part of the anterior cortex was absent and the tumor infiltrated into the soft tissues. Six years after the tibial resection, the patient developed pulmonary metastasis and died 1 year later of tumor. A lung biopsy showed classic adamantinoma, basaloid pattern.

Pathology Light Microscopy Routine histological studies were performed on 5-μmthick sections after formalin fixation, decalcification, and staining with hematoxylin–eosin. Ultrastructural Pathology

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Adamantinoma and Mesenchymal and Epithelial Cells    25 Immunohistochemistry Immunohistochemistry for cytokeratins AE1/AE3, CAM5.2, 7, 19, 20 and cytokeratin 903 was performed using the NexES automated immunostainer and detection systems (Ventana Medical Systems, Tucson, Arizona). Formalin-fixed, paraffin-embedded, 5-μm-thick sections were deparaffinized and rehydrated. Protease epitope retrieval was performed by incubating the tissue sections with a protease 1 enzyme (Ventana Medical Systems) for all cytokeratins except CK903, which had heat-induced epitope retrieval by microwaving. All antibodies were from Ventana Medical Systems and were prediluted. Primary antibody was detected by application of a biotinylated secondary anti-mouse antibody. Slides were then counterstained with hematoxylin. Negative controls consisted of incubation of tissue sections with isotypematched immunoglobulins without primary antibody.

Results Light Microscopy Histologic sections of all 3 cases revealed classic adamantinoma. Case 1 showed a predominant tubular pattern with small gland-like spaces usually lined by a single layer of cuboidal epithelial cells (Figure 4). Some small tubular structures contained slit-like lumina, which were often difficult to identify as epithelial elements (Figure 4). Case 2 showed a predominant basaloid pattern with solid masses of tumor cells, which frequently showed irregular spaces between the peripheral and central cells (Figure 5). Some of the spaces had

Electron Microscopy Electron microscopy of the 3 cases was performed on fresh tissue samples double fixed in 3% glutaraldehyde and 1% osmic acid. Due to tissue sampling, the material obtained for EM from case 2 included mainly central areas of the basaloid epithelial masses and stromal cells. In case 3, only single cells and small groups of epithelial cells and abundant stromal tissue were available for study.

Figure 4  Photomicrograph of tubular pattern (case 1) showing gland-like structures lined by cuboidal epithelial cells. Original magnification, ×200.

Figure 3  Radiograph of the lower two-thirds of the tibia showing irregular cortex with intramedullary tumor containing lytic and sclerotic areas. There is focal soft tissue infiltration in the medial region of the tibia. © 2012 Informa Healthcare USA, Inc.

Figure 5  Basaloid pattern (case 2) composed of epithelial masses of cells containing spaces(s) that separate the peripheral from the central cells. Original magnification, ×200.

26    C. D. Sarita-Reyes et al. a ­c­yst-like appearance. Case 3 showed in the resected tibial specimen a basaloid pattern of adamantinoma with prominent osteofibrous dysplasia-like pattern, which was located in the distal region of the tumor. Reactive fibrous tissue and focal sclerosis was present in the proximal region of the tumor.

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Immunohistochemistry IH studies of the epithelial cells of the 3 cases revealed strong reactivity to cytokeratins AE1/AE3, CK19, and CK903, mainly in the basaloid and tubular epithelial structures. In addition, few isolated AE1/AE3 positive cells were seen in the stroma of cases 1 and 2. In case 3, numerous AE1/AE3 and CK19 and fewer CK903positive cells were seen in the areas of the osteofibrous dysplasia-like pattern, which frequently formed small cellular aggregates (Figure 6). CAM 5.2 was positive in few epithelial and stromal cells of case 1 (Table 1).

generally of cuboidal shape, the cell membrane contained hemidesmosomes, and there was a peripheral basal membrane, which was mostly continuous. Tonofilaments, usually perinuclear, were frequently seen in approximately 30–50% of the cells. The luminal surface of the cells, without basal membrane, was irregular and contained numerous microvilli that lacked glycocalix and core filaments (Figure 7)  [19]. Occasional rudimentary intercellular junctions were present, and very rare desmosomes were also noted. Some tubular spaces were small with slit-like lumina and were lined by low cuboidal cells, which contained few scattered tonofilaments (Figure  8). Besides the tubular or gland-like spaces, solid cord-like structures containing mostly plump epithelial cells were seen, and small spaces were identified between the cells containing microvilli, which represent rudimentary tubular lumina (Figure 9).

Electron Microscopy Epithelial Cells The tubular pattern of adamantinoma showed epithelial cells lining gland-like spaces. The cells were

Figure 6  Stromal component of case 3 (osteofibrous dysplasialike pattern) showing numerous keratin-positive spindle cells with small focal cellular aggregates seen at right (AE1/AE3 keratin stain). Original magnification, ×200.

Figure 7  Tubular pattern with conspicuous glandular spaces lined by cuboidal cells surrounded by continuous basement membrane (arrowheads). Numerous microvilli are seen on the inner cell surface and tonofilaments are present (arrows). Original magnification, ×8250.

Table 1  Cytokeratin expression of adamantinomas. Case No. Diagnosis AE1/AE3 CAM5.2 1 Classic adamantinoma EC + (few cells) EC + SC + (few cells) SC + (few cells) 2 Classic adamantinoma EC + – SC + (few cells) – 3 Classic adamantinoma with EC + prominent osteofibrous SC + (many cells) dysplasia-like pattern Note. +, expression; –, no expression; EC, epithelial cells; SC, spindle cells.

CK7 – – –

CK19 EC + SC – EC + SC – EC + SC + (many cells)

CK20 – – –

CK903 EC + SC – EC + SC – EC + SC + (few cells)

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Adamantinoma and Mesenchymal and Epithelial Cells    27

Figure 10  Basaloid pattern showing a group of central elongated epithelial cells that are separated by a space(s) from the peripheral cells, which are not shown in the EM micrograph. The central cells contain few tonofilaments (arrow) and focal partial basal membrane (arrowheads) and are distributed rather loose from each other with lack of cohesion. Intercellular electron dense material is present. Original magnification, ×6500. Inset: Histologic counterpart of the EM picture showing the central basaloid cells separated from the peripheral cells by irregular spaces (S). Original magnification, ×200.

Figure 8  Tubular pattern composed of narrow glandular spaces lined by slightly flattened cells with basement membrane on the stromal surface (arrowheads) and focal tonofilaments (arrows). Original magnification, ×8250. Inset: Histologic appearance of the tubular pattern with narrow lumina that resembles primitive capillary vessels. Original magnification, ×200.

Figure 9  Tubular cord-like structure lined mainly by plump epithelial cells, containing microvilli at right lower region with small space, which represents a rudimentary tubular lumen. Tonofilaments are present (arrow). Original magnification, ×12,500. Inset: Histologic appearance of the tubular cord-like structures which are often seen in the tubular pattern. Original magnification, ×200.

In the basaloid pattern, the central cells within the epithelial masses, which were located adjacent to the spaces described histologically (see Figure 5), were stellate and ovoid in shape and were loosely disposed. © 2012 Informa Healthcare USA, Inc.

They contained sparse tonofilaments and partial basal membrane, and cell junctions were extremely rare. Abundant intercellular electron-dense material was seen between the cells (Figure 10). Rare epithelial cells contained, besides tonofilaments, actin-like filaments, glycogen, and lipid droplets. In addition to the epithelial cells described in the tubular and basaloid patterns, other types of cells with epithelial features were occasionally seen within the stroma of the 3 adamantinomas: first, single spindled cells with tonofilaments and rare basement membrane (Figure 11); and, second, round to stellate cells with conspicuous cytoplasm, perinuclear tonofilaments, and cell processes that lacked cell junctions and basement membrane (Figure 12). There were also rare spindled epithelial cells containing desmosomes that were forming small aggregates surrounded by partial basal membrane (Figure 13). These epithelial cells probably represent the keratin-positive cell aggregates previously illustrated in Figure 6. Stromal Cells The stromal cells were a minor component of cases 1 and 2, where the epithelial cells predominated. In case 3, the stromal component was more conspicuous. Fibroblasts were the main stromal cells, showing mild to moderate amounts of rough endoplasmic reticulum, with occasional dilatation. These cells usually had thin and elongated nuclei, showed rare intercellular connections, and were separated from each other by amorphous electron-dense material or scattered collagen fibers. Some spindled cells contained rare glycogen and actin-like filaments consistent with myofibroblasts.

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28    C. D. Sarita-Reyes et al.

Figure 11  Two spindle, poorly differentiated epithelial cells from case 1 (left) and case 2 (right) that were identified within the fibroblastic stroma. They contain tonofilaments (arrows) and partial basement membrane. Original magnification, ×12,500.

Figure 13  Small group of poorly differentiated epithelial cells (case 1), some with tonofilaments (arrows), basement membrane, and a desmosome (arrowhead) located at the lower part of picture. Original magnification, ×8250. Inset: High-power view of desmosome. Original magnification, ×10,500.

Figure 12  Round to stellate epithelial cells from case 3 containing tonofilaments (arrows) and cell projections without cellular junctions or basement membranes. Original magnification, ×8250.

Discussion Immunohistochemically, the epithelial cells of our three cases of classic adamantinoma were reactive to AE1/AE3, CK19, and CK903 and to a lesser degree to CAM5.2 (Table 1). Hazelbag et al. [9] found that keratins 14 and 19 were present in all 22 adamantinomas studied by the authors, and keratins 5, 17, 7, and 13 were seen in lesser amounts.

Numerous EM articles of classic adamantinoma have been published in the literature [3–7,10–16] and based on their illustrations, most of the authors described the basaloid pattern. To our knowledge, there is only one EM report of a case that, based on the illustrations and description, appeared to represent a tubular or glandular pattern of adamantinoma [17]. In our ultrastructural observations, the tubular or gland-like pattern consisted of spaces of varying size lined by flattened to cuboidal cells. The cuboidal epithelial cells were surrounded at the periphery by a basement membrane that appeared to be continuous and separated them from the stroma. Microvilli were often present between the cells and along the inner cell membrane. The lining cells sometimes protruded or extended into the tubular lumina, as described by Eisenstein et  al.  [17] in their case of probably tubular adamantinoma. Tonofilaments, one of the most specific features of epithelial cells, were seen in approximately 30–50% of cells. Mori et al. [14] found tonofilaments in about one-third of the tumor cells of a basaloid pattern of adamantinoma. The tubular adamantinoma may also contain small tubular and cord-like structures with narrow lumina and sparse tonofilaments, which made them Ultrastructural Pathology

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Adamantinoma and Mesenchymal and Epithelial Cells    29 very difficult to be identified as epithelial cells; they represent poorly differentiated glands that resemble primitive capillary vessels at the light microscopic level [2]. We were not able to find in the tubular cells features of endothelial differentiation (such as Weibel Palade granules), as claimed by other authors who proposed a vascular origin for adamantinoma [20,21]. In the basaloid pattern, light microscopy shows that spaces of different sizes may separate the peripheral cells from the central epithelial aggregates [2]. Our EM observations suggest that the central epithelial cells appeared to be less differentiated than the peripheral cells: they had sparse tonofilaments and partial basement membrane, and they were loosely separated from each other. Conspicuous electron-dense material was observed between them. Mori et al. [14] described somewhat similar ultrastructural observations. Some authors failed to find basal membrane around what appeared to be central epithelial cells [10]. We feel that the irregular or “cystlike” spaces within the epithelial masses are not true cell-lined spaces. On the contrary, they appeared to be the result of cell discohesion that probably is related to poor epithelial differentiation. The stromal component of our cases contained mostly fibroblasts and infrequent myofibroblasts. These latter cells have been previously documented at the EM level [12,15–17]. An interesting EM finding was the presence within the stroma of spindled and round to stellate epithelial cells admixed with fibroblasts. The spindled epithelial cells have been illustrated previously in classic adamantinoma at the IH [2,22,23] and by one of the authors (GCS) at the EM levels [22]. These cells have a lesser degree of epithelial differentiation than the classic epithelial cells of adamantinoma. In some areas of the stroma we also identified small groups of these epithelial cells in close proximity to each other like in an attempt to form tubular or gland-like structures. The histogenesis of adamantinoma is not known. Based on our observations, we feel that the presence of single poorly differentiated epithelial cells side by side within the stromal fibroblasts suggests that they probably originated from mesenchymal cells and underwent a process of mesenchymal–epithelial transformation. These epithelial cells resemble stromal cells on routine light microscopy and can be recognized as epithelial cells only by EM and IH. Some authors referred to the spindle epithelial cells as hybrid mesenchymal– epithelial cells and suggest that they likely arise from the neighboring stromal fibroblasts or their precursors, and may represent the neoplastic component of the stroma  [16]. This cellular mesenchymal–epithelial blending also has been suggested in previous histologic studies by several authors [7,12,18] and also was noted by the current authors. In an important study, in 1997, Hazelbag et al. [18] evaluated the distribution of matrix components of adamantinoma by IH and found continuous basement membrane staining and strong tenascin activity in well-developed epithelial fields. The opposite © 2012 Informa Healthcare USA, Inc.

was found in areas with less distinct epithelial features or in clearly fibrous fields. The authors also noted that in osteofibrous dysplasia-like adamantinoma, isolated keratin-positive cells displayed pericellular staining of basement membrane [18]. All these features in classic adamantinoma, according to the authors, are in accordance with the model of mesenchymal–epithelial conversion  [18]. A similar resemblance was found by some authors in the development of epithelium from mesenchymal cells in synovial sarcoma [24]. Epithelial features have been also described in other bone and soft tissue sarcomas, such as sclerosing epithelioid fibrosarcoma, Ewing sarcoma, and malignant fibrous histiocytoma. These findings were interpreted as divergent epithelial differentiation by some authors [25]. It is not known if the stromal cells of adamantinoma are reactive or neoplastic. In favor of their being reactive is the fact that they have not been reported to be present at the site of metastases in adamantinoma [16,26] and DNA aneuploidy was not present in these cells  [26]. On the other hand, features suggestive of neoplastic potential are the presence in the stromal cells of trisomies of chromosomes 7, 8, and 12 by cytogenetic and FISH (fluorescence in situ hybridization) studies [1,16,27,28]. These chromosomal abnormalities are also present in osteofibrous dysplasia of bone [16,27,28], which, together with osteofibrous dysplasia-like adamantinoma, are considered to be part of the histologic spectrum and histogenetically related to classic adamantinoma [2,16,18,27–29]. We agree with other authors that classic adamantinoma contains a dual population of cells, mesenchymal and epithelial, and it is probable that these two cells originate from a common mesenchymal stem cell [15,16,18,30].

Acknowledgement This work was partially presented at the Annual Meeting of the United States–Canadian Academy of Pathology, March 2007, San Diego, California. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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