Analysis of stromal cells in osteofibrous dysplasia and adamantinoma of long bones

Adamantinoma of long bones and osteofibrous dysplasia are rare, osteolytic primary bone tumours of uncertain origin containing areas of fibrous and fibro-osseous proliferation. We investigated the nature of the stromal cells in adamantinoma of long bones and osteofibrous dysplasia, and determined cellular and molecular mechanisms of osteolysis in these tumours. Cell culture, molecular (RT-PCR, western blot) and immunohistochemical studies on cases of adamantinoma of long bones and of osteofibrous dysplasia were undertaken to determine the expression of epithelial, osteoblast and osteoclast markers. Ultrastructural and immunophenotypic studies on cultured adamantinoma and osteofibrous dysplasia stromal cells showed that these cells were mainly fibroblast-like with few cells expressing epithelial markers. Osteofibrous dysplasia but not adamantinoma cells expressed alkaline phosphatase. Both osteofibrous dysplasia and adamantinoma cells expressed the ostoclastogenic factors M-CSF and RANKL. Adamantinoma and osteofibrous dysplasia cells also expressed messenger RNA for osteocalcin, osteonectin, osteopontin, osterix and collagen type 1. Adamantinoma and osteofibrous dysplasia cells cultured alone on dentine slices were not capable of lacunar resorption, but in co-cultures with monocytes induced formation of osteoclast-like cells was observered. Cultured osteofibrous dysplasia and adamantinoma stromal cells show similar ultrastructural and immunophenotypic characteristics, and differentially express osteoblast markers. Promotion of osteoclastogenesis by stromal cells may contribute to osteolysis in adamantinoma of long bones and osteofibrous dysplasia.     

Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors

Podoplanin is known as a lymphatic marker because its expression is detected in lymphatic but not vascular endothelium. Podoplanin is also expressed in several normal tissues including osteocytes or osteoblasts. A systematic examination of the podoplanin expression was conducted in normal skeletal tissues and some bone tumor cell lines, and the diagnostic value determined in primary bone tumors. Podoplanin mRNA was expressed at a high level in bone marrow tissue and cartilage, and was upregulated with differentiation to osteoblasts in bone marrow cells. Strong podoplanin expression was seen in osteocytes, chondrocytes, and osteoblasts on immunohistochemistry. Podoplanin mRNA was expressed at a high level in several osteosarcoma and chondrosarcoma cell lines, whereas podoplanin was expressed at a low level in a Ewing's/primitive neuroectodermal tumor cell line. In the clinical samples, osteosarcomas (22/26) expressed podoplanin at various levels. In small cell osteosarcomas (2/2), podoplanin was expressed strongly, although the tissue samples included few diagnostic osteoids. Chondrosarcomas (10/10) expressed podoplanin strongly, and chondroblastomas (5/5) expressed podoplanin moderately, while podoplanin was absent or expressed at low levels in Ewing's sarcomas (0/5), chordomas (0/6) and giant cell tumors of bone (1/7). Therefore, podoplanin may be a sensitive immunohistochemical marker of osteogenic and chondrogenic bone tumors.     

Adamantinoma of tibia with predominant features of fibrous dysplasia--a case report.

We report a case of adamantinoma of the tibia resembling fibrous dysplasia. The patient was a 55-year-old male, and complained of pain in the right lower leg. Roentgenographs showed a well demarcated osteolytic lesion with small foci of calcification and septation within the diaphysis of the distal tibia. The cortex was partially disrupted. Histologically, initial biopsy specimen showed fibrous connective tissue and trabeculae of immature woven bone, strongly suggestive of fibrous dysplasia. The lesion recurred and the second biopsy revealed nests of spindle cells and tubular epithelial structures embedded in granulation type-fibrous tissue. Immunohistochemically, both the nests of spindle cells and the tubular structures gave a positive reaction for cytokeratin. The present case emphasizes once again that histological diagnosis of fibrous dysplasia of the tibia should be made carefully with exclusion of the possibility of adamantinoma.     

Dentine matrix protein 1 (DMP-1) is a marker of bone-forming tumours

Dentin matrix protein 1 (DMP-1) is highly expressed by osteocytes and is a non-collagenous matrix protein found in dentin and bone. In this study, we determined the expression of DMP-1 in mature and immature human bone and examined whether DMP-1 is useful in distinguishing osteoid/bone-forming tumours from other primary and secondary bone tumours. DMP-1 expression was immunohistochemically determined in paraffin sections of a wide range of benign and malignant primary bone tumours and tumour-like lesions (n?=?353). DMP-1 mRNA expression was also examined in osteosarcoma and fibrosarcoma cell lines as well as bone tumour specimens (n?=?5) using real-time PCR. In lamellar and woven bone, DMP-1 was expressed in the matrix around osteocyte lacunae and canaliculi; osteoblasts and other cell types in the bone were negative. Matrix staining of the osteoid and bone was seen in bone-forming tumours including osteoma, osteoid osteoma, osteoblastoma and osteosarcoma. DMP-1 staining was also seen in fibrous dysplasia, osteofibrous dysplasia and chondroblastoma and in reactive bone in solitary bone cysts and aneurysmal bone cysts. DMP-1 was not expressed in the tumour component of other bone neoplasms including Ewing sarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, giant cell tumour of bone and metastatic carcinoma. DMP-1 mRNA was expressed in osteosarcoma cell lines and tumour samples. DMP-1 is a matrix marker expressed around osteocytes in human woven and lamellar bone and is useful in identifying osteosarcoma and other bone-forming tumours.     

Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers.     

Fibrous dysplasia with degenerative atypia: a benign lesion potentially mistaken for sarcoma

Fibrous dysplasia is a benign disorder of bone in which proliferating fibrous tissue replaces the bony spongiosa. Cytologic atypia is generally not considered a feature of this proliferating tissue. We present a case of fibrous dysplasia with marked atypical nuclear changes consistent with degenerative or regressive changes. A 42-year-old man presented with an osteolytic lesion of the right iliac bone. Histologic study demonstrated a fibro-osseous lesion with woven bone trabeculae and bland-looking fibrous tissue. Several areas showed atypical cells with enlarged pleomorphic nuclei and bizarre features. There was no change in the nuclear-cytoplasmic ratio nor were mitotic figures identified. The differential diagnosis is discussed. When radiographic and other histologic findings suggest fibrous dysplasia, the atypical nuclear changes should not, by themselves, alter the diagnosis.     

Comparative study of fibrous dysplasia and osteofibrous dysplasia: histopathological, immunohistochemical, argyrophilic nucleolar organizer region and DNA ploidy analysis

Fibrous dysplasia and osteofibrous dysplasia are both benign fibro-osseous lesions of the bone. We retrospectively studied the clinicopathological findings in 90 cases of fibrous dysplasia and 17 cases of osteofibrous dysplasia. In these cases, the expression of proliferating cell nuclear antigen (PCNA) and the presence of argyrophilic nucleolar organizer regions (AgNOR), as well as DNA ploidy, were examined. The bones affected by fibrous dysplasia were the maxilla, femur and frontal bone. Osteofibrous dysplasia occurred exclusively in the tibia or fibula. The average age of patients with fibrous dysplasia (24.0 years) was higher than that of patients with osteofibrous dysplasia (12.9 years). Fibrous dysplasias were divided into four major histological subtypes: Pagetoid, Chinese alphabet, small bone and parallel bone. Bone lining cells, which are known as resting osteoblasts, were seen in some cases of fibrous dysplasia. Cartilage differentiation was not seen in osteofibrous dysplasia. PCNA expression was strongly positive in the nuclei of osteoblasts around the bone trabeculae in osteofibrous dysplasia, but negative in the nuclei of bone lining cells around the bone trabeculae in fibrous dysplasia. The number of AgNOR in osteofibrous dysplasia was slightly higher than that in fibrous dysplasia. Both fibrous dysplasia and osteofibrous dysplasia were diploid. These features suggest that fibrous dysplasia can be differentiated from osteofibrous dysplasia by anatomical site, patient age, histological appearance, cartilage differentiation and PCNA positivity. DNA content by image cytometry is not a useful tool for differentiating these two diseases.     

Erlotinib inhibits osteolytic bone invasion of human non-small-cell lung cancer cell line NCI-H292

Previous preclinical and clinical findings have suggested a potential role of epidermal growth factor receptor (EGFR) in osteoclast differentiation and the pathogenesis of bone metastasis in cancer. In this study, we investigated the effect of erlotinib, an orally active EGFR tyrosine kinase inhibitor (TKI), on the bone invasion of human non-small-cell lung cancer (NSCLC) cell line NCI-H292. First, we established a novel osteolytic bone invasion model of NCI-H292 cells which was made by inoculating cancer cells into the tibia of scid mice. In this model, NCI-H292 cells markedly activated osteoclasts in tibia, which resulted in osteolytic bone destruction. Erlotinib treatment suppressed osteoclast activation to the basal level through suppressing receptor activator of NF-κB ligand (RANKL) expression in osteoblast/stromal cell at the bone metastatic sites, which leads to inhibition of osteolytic bone destruction caused by NCI-H292 cells. Erlotinib inhibited the proliferation of NCI-H292 cells in in vitro. Erlotinib suppressed the production of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), IL-8, IL-11 and vascular endothelial growth factor (VEGF) in NCI-H292 cells. Furthermore, erlotinib also inhibited osteoblast/stromal cell proliferation in vitro and the development of osteoclasts induced by RANKL in vitro. In conclusion, erlotinib inhibits tumor-induced osteolytic invasion in bone metastasis by suppressing osteoclast activation through inhibiting tumor growth at the bone metastatic sites, osteolytic factor production in tumor cells, osteoblast/stromal cell proliferation and osteoclast differentiation from mouse bone marrow cells.     

Immunohistochemical examination for the distribution of podoplanin-expressing cells in developing mouse molar tooth germs

We recently reported the expression of podoplanin in the apical bud of adult mouse incisal tooth. This study was aimed to investigate the distribution of podoplanin-expressing cells in mouse tooth germs at several developing stages. At the bud stage podoplanin was expressed in oral mucous epithelia and in a tooth bud. At the cap stage podoplanin was expressed on inner and outer enamel epithelia but not in mesenchymal cells expressing the neural crest stem cell marker nestin. At the early bell stage nestin and podoplanin were expressed in cervical loop and odontoblasts. At the root formation stage both nestin and podoplanin were weakly expressed in odontoblasts generating radicular dentin. Podoplanin expression was also found in the Hertwig epithelial sheath. These results suggest that epithelial cells of developing tooth germ acquire the ability to express nestin, and that tooth germ epithelial cells maintain the ability to express podoplanin in oral mucous epithelia. The expression of podoplanin in odontoblasts was induced as tooth germ development advanced, but was suppressed with the completion of the primary dentin, suggesting that podoplanin may be involved in the cell growth of odontoblasts. Nestin may function as an intermediate filament that binds podoplanin in odontoblasts.     

Podoplanin as a marker for mesothelioma

Podoplanin is a specific marker for lymph vessel endothelial cells. It was noted that podoplanin is expressed in reactive mesothelial cells. The utility of podoplanin for the histological diagnosis of tumors was then investigated, especially for mesothelioma. Immunohistochemical study of podoplanin was carried out in five malignant mesotheliomas and 118 other tumors including 93 adenocarcinomas, four squamous cell carcinomas, six gastrointestinal stromal tumors and five endocrine tumors. Immunoreactivity for podoplanin was demonstrated on the cell membrane of tumor cells for all mesotheliomas. All other tumors were negative for podoplanin. Among the many antibodies used for differential diagnosis of malignant mesothelioma, podoplanin has the potential to be an excellent tumor marker in both specificity and sensitivity. The utility of podoplanin as a marker for mesothelioma will be confirmed by further studies.     

Osteofibrous dysplasia: two affected male sibs and an unrelated girl with bilateral involvement

Osteofibrous dysplasia (OFD) is a tumor-like bone lesion that occurs most often in the tibia, presenting as a painless swelling or anterior bowing. Radiographs show a well-circumscribed intracortical lucency, or multiple lucencies separated by sclerotic borders, associated with a diaphyseal expansion. The histogenesis of OFD and its possible relationships to fibrous dysplasia and to adamantinoma have been the subject of significant discussion and investigation. We have been unable to find any reports of familial OFD, and have found only two references to bilateral involvement. In this article, we report both bilateral and familial involvement, and suggest that more thorough investigation of patients and their families may uncover similar cases, and perhaps support a genetic component to the etiology of this condition.     

Smooth muscle actin expression in primary bone tumours

Alpha isoform of smooth muscle actin (SMA) expression has been reported in giant cell tumour of bone (GCTB) and other benign and malignant bone tumours, but the pattern of SMA expression and the precise nature of SMA-expressing cells in these lesions is uncertain. We determined by immunohistochemistry the expression of SMA and other muscle and vascular markers in normal bone, GCTB and a wide range of primary benign and malignant bone tumours. Cultured stromal cells of GCTB, chondroblastoma (CB), and aneurysmal bone cyst (ABC) were also analysed for SMA expression. SMA was only noted in blood vessels in normal bone. SMA was expressed by mononuclear stromal cells (MSC) cultured from GCTB, ABC and CB. SMA was strongly and diffusely expressed by MSC in non-ossifying fibroma, fibrous dysplasia, and "brown tumour" of hyperparathyroidism. SMA expression was also noted in GCTB, ABC, CB, chondromyxoid fibroma, malignant fibrous histiocytoma of bone and osteosarcoma. Little or no SMA was noted in Langerhans cell histiocytosis, simple bone cyst, Ewing's sarcoma, osteoblastoma, osteoid osteoma, enchondroma, osteochondroma, chondrosarcoma, myeloma, lymphoma, chordoma and adamantinoma. Our findings show that there is differential SMA expression in primary bone tumours and that identifying the presence or absence of SMA is useful in the differential diagnosis of these lesions. The nature of SMA-expressing cells in bone tumours is uncertain but they are negative for desmin and caldesmon and could represent either myofibroblasts or perivascular cells, such as pericytes.     

Osteofibrous dysplasia and adamantinoma: correlation of proto-oncogene product and matrix protein expression

To investigate the relationship between osteofibrous dysplasia (OFD) and adamantinoma, we analyzed the expression of several proto-oncogene products and extracellular matrix proteins by immunohistochemistry and correlated our results with histological and ultrastructural findings. C-fos and c-jun, but not c-Met, were observed in OFD and in the fibrous and epithelial components of differentiated and classical adamantinomas. Staining for collagen IV, laminin and galectin-3, a laminin binding protein was seen in OFD and around cell nests in adamantinoma. E-, P-, and N-cadherin expression was found in all cases of classical adamantinoma, but not in differentiated adamantinoma or OFD. Osteonectin was detected in both the epithelial and fibrous components of adamantinomas, but osteopontin and osteocalcin were not seen in classical adamantinomas. The results show common expression of a number of oncoproteins and bone matrix proteins in adamantinoma and OFD, some of which are associated with mesenchymal-to-epithelial cell transformation. These findings would be in keeping with the hypothesis that OFD represents a precursor lesion of adamantinoma. Differential expression of a number of bone matrix protein in adamantinoma may also be of diagnostic use in distinguishing these 2 lesions immunohistochemically.     

Diffuse membranous immunoreactivity for podoplanin (D2-40) distinguishes primary and metastatic seminomas from other germ cell tumors and metastatic neoplasms

Podoplanin has been shown to be expressed in primary germ cell tumors (GCTs), with conflicting results regarding its specificity. However, podoplanin expression in metastatic GCTs and other metastatic tumors has not been extensively examined. The goal of this study was to evaluate the distribution and specificity of podoplanin using monoclonal antibody D2-40 in primary testicular and metastatic GCTs in comparison with other metastatic neoplasms. In total, 122 tumors were studied: 43 primary GCTs, 33 metastatic GCTs, 11 metastatic melanomas, 25 metastatic carcinomas, and 10 lymphomas. All foci of seminoma showed strong, diffuse membranous staining in more than 90% of cells in primary and metastatic GCTs. In contrast, other GCT components showed only focal cytoplasmic and/or partial membranous staining in a subset of cases. Among non-GCTs, only 1 metastatic melanoma, 1 lymphoma, and 3 metastatic carcinomas showed focal, weak cytoplasmic staining. Diffuse membranous immunoreactivity for podoplanin as detected by monoclonal antibody D2-40 is highly sensitive and specific for primary and metastatic seminoma. Immunodetection of podoplanin may be useful to support seminoma in the differential diagnosis of poorly differentiated epithelioid malignant neoplasms.     

Podoplanin is a useful marker for identifying mesothelioma in malignant effusions

The diagnosis of malignant mesothelioma in serosal effusions continues to be a major challenge because some of its cytomorphological features closely resemble adenocarcinomas. Immunohistochemistry is a valuable tool in the differentiation of epithelioid mesothelioma from metastatic adenocarcinomas. However, no single antibody has demonstrated absolute sensitivity or specificity. In this study, we evaluated the value of immunostaining pattern for podoplanin to differentiate mesothelioma from adenocarcinomas of various origins. Cell blocks from previously collected paraffin‐embedded cell blocks of 86 effusions (18 mesothelioma, 35 reactive mesothelium, 9 breast adenocarcinoma, 14 ovarian adenocarcinoma, and 10 lung adenocarcinoma) were retrieved from the file of the Department of Pathology at University of Michigan and Lund University in Sweden and were used for the study. Slides prepared from the cell blocks were stained for podoplanin. The percentage of immunostained cells was recorded as follows: 1+ (5–25%), 2+ (26–50%), and 3+ (>50%). A stain result involving <5% of cells was considered negative. The intensity of positive results was evaluated as strong, moderate, or weak. Podoplanin is expressed in 94% of malignant mesothelioma cases (17/18), 97% (30/31) of cases of reactive mesothelial, 0% of lung adenocarcinoma cases (0/9), 0% of breast adenocarcinoma (0/9), and 7% of ovarian adenocarcinoma (1/14). All positive cases of malignant mesothelioma and reactive mesothelium showed strong membranous reactivity to podoplanin. The one positive case of ovarian adenocarcinoma showed a weak membranous podoplanin immunostaining. On the basis of our results and published data, we believe that membranous podoplanin immunoreactivity, in conjunction with calretinin, would be more specific than CK5/6 and WT‐1 in differentiating epithelioid malignant mesothelioma from adenocarcinoma of the lung, breast, and ovary. Diagn. Cytopathol. 2010. © 2010 Wiley‐Liss, Inc.     

The expression of podoplanin and classic cadherins in the mouse brain

Podoplanin is a transmembrane glycoprotein indirectly linked to classic cadherins through ezrin-actin networks. Recently, the overexpression of podoplanin in high-grade malignancy brain tumors has been reported. The aim of this study was to investigate the expression of podoplanin and classic cadherins in the mouse brain. Immunohistochemistry showed that podoplanin was expressed on ependymal cells and choroid plexus epithelial cells at the ventricle side of the cell surface and at the cell-cell junctions, and on retinal pigment epithelial cells and in the pia mater; P-cadherin between choroid plexus epithelial cells and endothelial cells at the basement membrane side of cell surface, and between retinal pigment epithelial cells; VE-cadherin on the PECAM-1 positive-choroid plexus endothelial cells of the fibrovascular core; and N-cadherin on the cell surface and at the cell-cell junctions of ependymal cells, and in the pia mater. The regions expressing podoplanin, P-cadherin, and VE-cadherin did not coincide. In real-time PCR analysis, the amounts of podoplanin and P- and N-cadherin mRNA were larger in the ventricular wall with choroid plexus than in the abdominal aorta and cerebrum. In the RT-PCR analysis, the intensities of amplicon for VE-cadherin mRNA were the same for the abdominal aorta, cerebrum, and ventricular wall with the choroid plexus, suggesting that mouse ependymal cells, choroid plexus epithelial cells, and glial cells under the pia mater have the ability to express podoplanin and P- and N-cadherins. Glial cells and retinal pigment epithelial cells may create barriers by podoplanin and classic cadherins as a rate-determining step for transmission of blood components.     

Posttraumatic fibro-osseous lesion of rib.

Eleven cases are described of an unusual, benign, fibro-osseous lesion of rib previously reported under a variety of designations, including painless fibro-osseous lesion resembling osteoid osteoma, symmetrical fibro-osseous dysplasia, focal Erdheim-Chester disease, and fibro-osseous pseudotumor. All patients were adults, most of whom were asymptomatic, the lesion discovered by bone scans done to rule out metastatic disease. A single rib was involved in eight patients and multiple ribs in three. A roentgenographic abnormality was apparent in only five patients. Histologically, all lesions showed a bland fibrous stroma in which resided an anastomosing network of bone trabeculae, having a zonal pattern of maturation from metaplastic woven to mature lamellar bone, with or without an associated xanthomatous component. Seven patients had a history of previous trauma, three with fractured ribs. Considering the relative infrequency of solitary rib lesions attributable to metastatic disease, it is proposed that in most cases there is no need for a diagnostic rib resection for these incidentally discovered, posttraumatic reparative lesions.     

In vivo leptin expression in cartilage and bone cells of growing rats and adult humans

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin-embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.     

Ossifying fibroma vs fibrous dysplasia of the jaw: molecular and immunological characterization.

Ossifying fibroma and fibrous dysplasia of the jaw are maxillofacial fibro-osseous lesions that should be distinguished each other by a pathologist because they show distinct patterns of disease progression. However, both lesions often show similar histological and radiological features, making distinction between the two a diagnostic dilemma. In this study, we performed immunological and molecular analyses of five ossifying fibromas, four cases of extragnathic fibrous dysplasia, and five cases of gnathic fibrous dysplasia with typical histological and radiographic features. First, we examined the difference between fibrous dysplasia and ossifying fibroma in the expression of Runx2 (which determined osteogenic differentiation from mesenchymal stem cells) and other osteogenic markers. Fibroblastic cells in fibrous dysplasia and ossifying fibroma showed strong Runx2 expression in the nucleus. The bone matrices of both lesions showed similar expression patterns for all markers tested except for osteocalcin. Immunoreactivity for osteocalcin was strong throughout calcified regions in fibrous dysplasia, but weak in ossifying fibroma lesions. Second, we performed PCR analysis with peptide nucleic acid (PNA) for mutations at the Arg(201) codon of the alpha subunit of the stimulatory G protein gene (GNAS), which has reported to be a marker for extragnathic fibrous dysplasia. All nine cases of extragnathic or gnathic fibrous dysplasia were positive for this mutation. On the other hand, none of the five cases of ossifying fibroma showed the mutation. These findings indicate that although fibrous dysplasia and ossifying fibroma are similar disease entities, especially in the demonstration of the osteogenic lineage in stromal fibroblast-like cells, they show distinct differences that can be revealed by immunohistochemical detection of osteocalcin expression. Furthermore, PCR analysis with PNA for GNAS mutations at the Arg(201) codon is a useful method to differentiate between fibrous dysplasia and ossifying fibroma.