Adamantinoma-Like Ewing Sarcoma Mimicking Merkel Cell Carcinoma in the Parotid Gland: A Diagnostic Pitfall.

Adamantinoma-like Ewing sarcoma (ALES) is a newly described rare entity, which shows EWSR1::FLI1 rearrangement characteristic of Ewing sarcoma. This can be diagnostically challenging as it manifests histologically with epithelial differentiation and has diffuse keratin expression as well as p40 and p60 positivity. We hereby report a case of ALES in a 33-year-old woman with a past medical history of breast carcinoma who presented with a right-sided parotid mass. CT scan of the neck showed a heterogenous mass within the superficial lobe, measuring 17 mm in diameter for which the patient underwent superficial parotidectomy. Histopathology of the mass revealed a malignant neoplasm formed of solid nests, cords and sheets of cells with minimal cytoplasm and monomorphic nuclei with granular chromatin and indistinct nucleoli. Brisk mitotic activity and tumor necrosis were also present. The tumor showed strong and diffuse reactivity for pankeratin (clone AE1/AE3) and keratin 20, both in a dot-like pattern, raising the suspicion of metastatic Merkel cell carcinoma; however, molecular studies showed EWSR1::FLI1 rearrangement, supporting the diagnosis of ALES. In summary, it is prudent to have knowledge about this entity to avoid its misdiagnosis as other malignancies of the head and neck region which exhibit a different clinical course, prognosis and hence treatment modalities.

DNA Methylation Profiling Distinguishes Adamantinoma-Like Ewing Sarcoma From Conventional Ewing Sarcoma.

Adamantinoma-like Ewing sarcoma (ALES) has traditionally been considered a variant of Ewing sarcoma because it generally harbors EWSR1::FLI1 fusions despite showing diffuse positivity for keratins and p40. However, it has become increasingly recognized that different tumors can have identical translocations, including shared fusions between carcinomas and sarcomas, raising questions as to whether ALES might represent a separate entity. Using methylation profiling, we further explored the relationship between Ewing sarcoma and ALES. The archives of multiple institutions were searched for candidate cases of ALES. DNA methylation profiling was performed and results were compared to corresponding data from conventional Ewing sarcoma. Twelve cases of ALES (5 previously reported) were identified in 10 men and 2 women (aged 20-72 years; median age, 41.5 years). Cases included tumors arising in the parotid gland (3), sinonasal cavity (2), submandibular gland (2), thyroid gland (1), neck (1), gingiva (1), hypopharynx (1), and mandible (1). Histologic review consistently showed sheets and nests of basaloid cells within a fibromyxoid or hyalinized stroma. All tumors were positive for at least 1 keratin and CD99 expression, whereas all 10 cases tested were positive for p63 or p40; S100 protein expression was noted in 2 cases. Cases harbored either EWSR1::FLI1 fusions (n = 6), FUS::FLI1 fusions (n = 1), and/or EWSR1 rearrangements (n = 6). Methylation profiling was successful in 11/12 cases evaluated. Unsupervised clustering and dimensionality reduction (Uniform Manifold Approximation and Projection) of DNA methylation data revealed a distinct methylation cluster for all 11 cases, including the tumor with the FUS::FLI1 fusion, which clearly segregated them from the conventional Ewing sarcoma. Follow-up (n = 11, 1-154 months) revealed that 4 patients experienced recurrence and 6 developed metastatic disease. ALES demonstrates a distinct methylation signature from conventional Ewing sarcoma. This finding adds to the distinctive immunoprofile of ALES, suggesting that these 2 tumors should be considered distinct entities rather than histologic extremes of the same disease.

Adamantinoma-like Ewing Sarcoma (ALES) May Harbor FUS Rearrangements : A Potential Diagnostic Pitfall.

Adamantinoma-like Ewing sarcoma (ALES) is a rare malignancy currently considered a variant of Ewing sarcoma with most known cases harboring EWSR1 rearrangements. Herein we present a series of 6 cases of EWSR1 -negative ALES. The tumors arose in the sinonasal tract (n=3), major salivary glands (submandibular gland=1; parotid=1), and anterior mediastinum (n=1) in patients ranging from 25 to 79 years of age. Most tumors were basaloid in appearance, growing in large nests separated by interlobular fibrosis without overt squamous pearls. However, 1 case closely resembled a well-differentiated neuroendocrine tumor with uniformly round nuclei, eosinophilic cytoplasm, and trabecular architecture. All cases were diffusely positive for pan-cytokeratin, p40 or p63, and CD99. A subset of cases showed diffuse reactivity for synaptophysin, including 1 sinonasal tumor which also demonstrated sustentacular S100 protein expression. Molecular testing showed FUS rearrangements in all cases. Gene partners included known ETS family members FEV (n=2) and FLI1 (n=1). Our results expand the molecular diagnostic considerations for ALES to include FUS rearrangements. We also show that ALES may harbor FUS :: FLI1 fusion, which has not been previously reported in the Ewing family of tumors. Furthermore, ALES may show unusual histologic and immunophenotypic features that can overlap with olfactory carcinoma including S100-positive sustentacular cells. ALES should be considered in the diagnostic differential of small round cell tumors and tumors with neuroendocrine differentiation with immunohistochemical workup to include p40 and CD99/NKX2.2.

Transcriptomic and immunophenotypic characterization of two cases of adamantinoma-like Ewing sarcoma of the thyroid gland.

INTRODUCTION: Adamantinoma-like Ewing sarcoma (ALES) is a rare aggressive malignancy occasionally diagnosed in the thyroid gland. ALES shows basaloid cytomorphology, expresses keratins, p63, p40, frequently CD99, and harbours the t(11;22) EWSR1::FLI1 translocation. There is debate on whether ALES resembles more sarcoma or carcinoma. METHODS: We performed RNA sequencing from two ALES cases and compared findings with skeletal Ewing's sarcomas and nonneoplastic thyroid tissue. ALES was investigated by in situ hybridization (ISH) for high-risk human papillomavirus (HPV) DNA and immunohistochemistry for the following antigens: keratin 7, keratin 20, keratin 5, keratins (AE1/AE3 and CAM5.2), CD45, CD20, CD5, CD99, chromogranin, synaptophysin, calcitonin, thyroglobulin, PAX8, TTF1, S100, p40, p63, p16, NUT, desmin, ER, FLI1, INI1, and myogenin. RESULTS: An uncommon EWSR1::FLI transcript with retained EWSR1 exon 8 was detected in both ALES cases. Regulators of EWSR1::FLI1 splicing (HNRNPH1, SUPT6H, SF3B1) necessary for production of a functional fusion oncoprotein, as well as 53 genes (including TNNT1, NKX2.2) activated downstream to the EWSR1::FLI1 cascade, were overexpressed. Eighty-six genes were uniquely overexpressed in ALES, most of which were related to squamous differentiation. Immunohistochemically, ALES strongly expressed keratins 5, AE1/AE3 and CAM5.2, p63, p40, p16, and focally CD99. INI1 was retained. The remaining immunostains and HPV DNA ISH were negative. CONCLUSION: Comparative transcriptomic profiling reveals overlapping features of ALES with skeletal Ewing's sarcoma and an epithelial carcinoma, as evidenced by immunohistochemical expression of keratin 5, p63, p40, CD99, the transcriptome profile, and detection of EWSR1::FLI1 fusion transcript by RNA sequencing.

Adamantinoma with a Prominent Spindle Cell Component Mimicking Intraosseous Synovial Sarcoma: Clinicopathological Features of Six Tumors.

Introduction. Adamantinoma is sub-classified into classic/biphasic, osteofibrous dysplasia-like, and de-differentiated type. We present six adamantinomas with a prominent spindle cell component mimicking intraosseous synovial sarcomas. Methods. Six patients were either referred with a diagnosis of intraosseous synovial sarcoma or wherein synovial sarcoma was a differential diagnosis. Three tumors were tested for SS18 gene rearrangement by FISH and two for SS18::SSX fusion by RT-PCR technique. Results. There were three males and three females with an average age of 20.6 years. Radiologically, the lesions were expansile and showed lytic and/or sclerotic components, involving the cortex and/or medulla. Five lesions occurred in the tibia and two in the fibula. Two tumors displayed soft tissue extension and two occurred as multifocal lesions. Two patients were diagnosed with synovial sarcoma and a single patient with sarcomatoid carcinoma, elsewhere. Two "in-house" patients were initially diagnosed with synovial sarcomas. On review, all tumors were cellular comprising monomorphic spindle-shaped cells arranged in sheets and fascicles (n = 6), including a "herringbone-like" pattern (n = 3), focal tubules (n = 1), cohesive nests (n = 5), cords (n = 2), including pseudocystic component (n = 2). Immunohistochemically, tumor cells were positive for p63 (6/6), p40 (4/4), EMA (2/3), AE1/AE3 (5/6), various keratins (2/2), and TLE1 (2/4). Three tumors tested for SS18 rearrangement were negative, while two tumors tested for SS18::SSX fusion were negative. Conclusions. Adamantinomas with spindle cell morphology display overlapping features with synovial sarcoma. A clinico-radiological index of suspicion immunostains (p63 and p40) and molecular test for t(X; 18) translocation are useful in an exact diagnosis, which has treatment-related implications.

Desmoplastic Adamantinoma-like Thymic Carcinoma: Clinicopathologic, Immunohistochemical, and Molecular Study of 5 Cases.

Five cases of a heretofore unreported rare variant of thymic carcinoma characterized by a striking resemblance to adamantinoma of the mandible are described. The tumors occurred in 4 women and 1 man aged 58 to 76 years (mean: 67.8 y); they arose in the anterior mediastinum and measured from 5.3 to 12.0 cm in greatest diameter (mean: 8.9 cm). Presenting symptoms included chest pain, shortness of breath, and in 2 patients, pleural effusion. One tumor was asymptomatic and discovered incidentally. Histologically, the tumors were extensively desmoplastic, and the cellular proliferation was characterized by multiple islands of squamous epithelium with striking peripheral palisading of nuclei and central areas containing clear cells resembling a stellate reticulum. Areas of preexisting spindle cell thymoma were identified in 2 cases; these areas gradually merged with the higher-grade component of the lesion. Cystic changes were noted in 3 cases. Immunohistochemical studies in 3 cases showed the tumor cells were positive for cytokeratins, p40 and p63, and all showed a high proliferation rate (>50% nuclear positivity) with Ki-67. Next-generation sequencing was performed in 2 cases that showed amplification of the AKT1 gene (copy numbers 6 and 13). Clinical follow-up in 3 patients showed recurrence and metastasis after 1 and 2 years; 1 patient passed away 2 years after diagnosis due to the tumor. Desmoplastic adamantinoma-like thymic carcinoma represents an unusual histologic variant of thymic carcinoma that needs to be distinguished from metastases from similar tumors to the mediastinum.

Outcome for Neoadjuvant Treatment of Parotid Gland Adamantinoma-Like Ewing Sarcoma: Case Report and Review of Literatures.

Adamantinoma-like Ewing sarcoma typically shows t(11;22) EWSR1::FLI1 translocation and complex epithelial differentiation. It poses a diagnostic challenge, especially in the head and neck region, due to its under-recognition and significant histologic overlap with other malignancies. Neoadjuvant and adjuvant treatment information on head and neck Adamantinoma-like Ewing sarcoma is limited. Herein, we report a case of a 78-year-old female with Adamantinoma-like Ewing sarcoma of the parotid gland, including the imaging findings and clinical response to neoadjuvant therapy followed by surgery. The efficacy of neoadjuvant therapy in the treatment of Adamantinoma-like Ewing sarcoma is discussed in the context of a review of pertinent literature. Adamantinoma-like Ewing sarcoma in the head and neck is frequently misdiagnosed as poorly differentiated squamous cell carcinoma or a basaloid salivary gland carcinoma. Adamantinoma-like Ewing sarcoma is a EWS1::FLI1 translocation driven tumor; frequently misdiagnosed on head and neck biopsies as poorly differentiated carcinoma, or squamous cell carcinoma. Ewing sarcoma-specific chemoregimen appears effective for this entity. If diagnosed early, patient may be amenable to neoadjuvant therapy, which may improve surgical and cosmetic outcomes. This is especially important in head and neck regions.

A unique case of adamantinoma-like Ewing sarcoma in the calcaneus, exhibiting prominent squamous differentiation and displaying EWSR1 gene rearrangement.

Adamantinoma-like Ewing sarcoma is uncommonly reported in the skeletal sites, including small bones of the feet.A 15-year-old girl presented with pain and swelling in her left foot, leading to difficulty in walking for 8 months. Plain radiograph revealed an ill-defined, lytic-sclerotic lesion without significant periosteal reaction in her left calcaneus. Magnetic resonance imaging (MRI) revealed an expansile lesion involving the anterior calcaneus, which was hypointense on T1 and heterogeneously hyperintense on T2-weighted sequences, infiltrating the adjacent bones and soft tissues. On imaging, the differential diagnoses considered were a giant cell tumor and other primary bone tumors.Histopathological examination revealed a tumor composed of small round cells, with interspersed keratin pearls. Immunohistochemically, the tumor cells were positive for CD99/MIC2, pan-cytokeratin (AE1/AE3), p40, p63, NKX2.2, and synaptophysin. Diagnosis of adamantinoma-like Ewing sarcoma was offered on the initial biopsy. Furthermore, the tumor cells revealed EWSR1 gene rearrangement by fluorescence in situ hybridization, confirming this diagnosis. The patient underwent neoadjuvant chemotherapy, had a poor response, and finally underwent below-knee amputation.This constitutes a rare case of adamantinoma-like Ewing sarcoma in the calcaneus. Ewing sarcoma may be considered as a differential diagnosis for intraosseous lytic-sclerotic lesions, even without significant periosteal reaction, at unusual sites, such as the bones of the foot. Awareness of this entity and application of ancillary techniques is recommended for its exact diagnosis and in differentiating this rare variant from its diagnostic mimics. This case also indicates a poor chemotherapy response in this unusual variant of Ewing sarcoma, occurring in the calcaneus.

Adamantinoma-like Ewing sarcoma of the thyroid.

Adamantinoma-like Ewing sarcoma (ALES) is a rare variant of Ewing sarcoma family of tumors (ESFTs). ALES is characterized by translocations of the EWSR1 (Ewing sarcoma breakpoint region 1) gene on chromosome 22, morphologic features of small round blue cell tumors with focal squamous differentiation, and a unique immunoprofile. Herein, we report a 21-year-old patient who presented with severe, acute onset back pain. Imaging revealed a large, exophytic, heterogeneously enhancing mass in the left thyroid and numerous lytic bone lesions. Fine-needle aspiration of the thyroid, revealed a cellular smear with "small round blue cell" morphology. The unique immunoprofile of positive ESFT markers (NKX2.2 and CD99), along with positive markers of squamous epithelial differentiation (AE1/AE3 and p40), led to a diagnosis of ALES. This was confirmed by fluorescence in situ hybridization, which demonstrated EWSR1 rearrangement in 74% of nuclei.

Comprehensive Molecular Characterization of Adamantinoma and OFD-like Adamantinoma Bone Tumors.

Adamantinoma and osteofibrous dysplasia (OFD)-like adamantinoma are rare primary bone tumors that are predominantly confined to the tibia. These 2 entities show similarities in location, histology, and radiologic appearance; however, adamantinoma is malignant and therefore differentiating between these bone tumors is essential for optimal patient care. To elucidate their genomic and transcriptomic alteration profiles and expand their etiological mechanisms, whole exome sequencing (WES) and RNA sequencing (RNA-Seq) were conducted on adamantinoma and OFD-like adamantinoma tumors. Copy number variation analysis using WES data revealed distinct chromosomal alteration profiles for adamantinoma tumors compared with OFD-like adamantinomas, allowing molecular differentiation between the 2 tumor subtypes. Combining WES and copy number variation analyses, the chromatin remodelling-related gene KMT2D was recurrently altered in 3/8 adamantinoma tumors (38%), highlighting the potential involvement of deregulated chromatin structure and integrity in adamantinoma tumorigenesis. RNA-Seq analysis revealed a novel somatic gene fusion (EPHB4-MARCH10) in an adamantinoma, the gene fusion was fully characterized. Hierarchical clustering analysis of RNA-Seq data distinctly clustered adamantinoma tumors from OFD-like adamantinomas, allowing to molecularly distinguish between the 2 entities. David Gene Ontology analysis of differentially expressed genes identified distinct altered pathways in adamantinoma and OFD-like adamantinoma tumors, highlighting the different histopathologic characteristics of these bone tumor subtypes. Moreover, RNA-Seq expression profiling analysis identified elevated expression of DLK1 gene in adamantinomas, serving as a potential molecular biomarker. The present study revealed novel genetic and transcriptomic insights for adamantinoma and OFD-like adamantinoma tumors, allowing to differentiate genetically and transcriptomically between the 2 lesions and identifying a potential diagnostic marker for adamantinomas.

Adamantinoma-Like Ewing Sarcoma of the Thyroid: A Case Report and Review of the Literature.

Currently considered a variant of Ewing sarcoma, adamantinoma-like Ewing sarcoma is a rare malignancy that shows classic Ewing sarcoma-associated gene fusions but also epithelial differentiation. Here we present the 6th reported case of adamantinoma-like Ewing sarcoma involving the thyroid gland. Sections of the thyroid tumor from a 20-year old woman showed sheets, lobules and trabeculae of primitive, uniform, small round blue cells that diffusely expressed pankeratin, p40 and CD99. Fluorescent in situ hybridization revealed an EWSR1 gene rearrangement and an EWSR1-FLI1 fusion was detected by RT-PCR. Neck lymph nodes were not involved, and the patient was treated with a Ewing sarcoma chemotherapy protocol and radiation and is disease free 7 months after surgery. The unusual histology and immunohistochemical profile of adamantinoma-like Ewing sarcoma makes diagnosis and classification very challenging. We also present a literature review of adamantinoma-like Ewing sarcoma involving the thyroid.

Genetic and molecular reappraisal of spindle cell adamantinoma of bone reveals a small subset of misclassified intraosseous synovial sarcoma.

Adamantinoma represents a distinct group of bone tumors showing both mesenchymal and epithelial differentiation most commonly involving the tibial diaphysis. Most adamantinomas contain a fibro-osseous component and an epithelial component consisting of squamous or basaloid cells. Adamantinomas are considered malignant neoplasms requiring en bloc excision that frequently recur locally and can rarely metastasize. Rare adamantinomas show an epithelial component consisting predominantly of monomorphic spindle cells, which, combined with an epithelial immunophenotype, can mimic monophasic synovial sarcoma. Synovial sarcoma is very rare in bone. It is considered a high-grade sarcoma that typically necessitates chemotherapy. However, the relationship between spindle cell adamantinoma and intraosseous synovial sarcoma has not been investigated. The current study was prompted by identification of a presumed spindle cell adamantinoma of the tibia with diffuse keratin expression that harbored a SS18 gene region rearrangement. FISH of eight additional bone tumors initially classified as spindle cell adamantinoma based on clinicoradiopathologic findings revealed one additional case with SS18 rearrangement. Histologically, both intraosseous synovial sarcoma and spindle cell adamantinoma demonstrated uniform fusiform nuclei with scant cytoplasm, short fascicles and low mitotic activity. The adamantinomas, but not the synovial sarcomas, were more likely to show overt epithelial differentiation in the form of pseudoglands or squamous nests. Immunohistochemistry of all cases, irrespective of SS18 status, showed diffuse keratin positivity in the spindle cell component, and less consistent EMA positivity. Clinical follow-up was available in both intraosseous synovial sarcomas, one of which recurred and the other metastasized. Two of the six spindle cell adamantinomas with follow-up metastasized. The above findings highlight the morphologic and immunophenotypic overlap between spindle cell adamantinoma and intraosseous synovial sarcoma of the tibia. Investigation of SS18 status to exclude synovial sarcoma is suggested prior to rendering a diagnosis of spindle cell adamantinoma.

Sinonasal adamantinoma-like Ewing sarcoma: A case report.

We describe the case of a sinonasal adamantinoma-like Ewing sarcoma in a 41-year-old male. Histologically, the tumor exhibited distinctive areas of nested growth pattern with prominent stromal fibrosis and metaplastic bone formation. The tumor cells were small and uniform with minimal amount of pale eosinophilic to clear cytoplasm and round or oval nuclei with finely dispersed chromatin and small nucleoli. Approximately 20% of the tumor parenchyma comprised of small clusters of basaloid cells within an osteofibrous background resembling adamantinoma. The tumor showed strong expression of keratins, p63, CD99 and Fli-1, and EWSR1 rearrangement. The diagnosis of sinonasal Ewing family tumors is particularly problematic owing to the large number of potential mimics. For any poorly differentiated or undifferentiated head and neck tumor, cellular monotony and CD99 immunoreactivity should prompt consideration for molecular studies that include analysis of both EWSR1 and FLI1, even in the presence of strong cytokeratin expression or focal keratinization.

Clinicopathological features of five unusual cases of intraosseous myoepithelial carcinomas, mimicking conventional primary bone tumours, including EWSR1 rearrangement in one case.

Primary intraosseous myoepithelial tumours, including carcinomas are rare tumours. The concept of histopathological spectrum of these tumours is evolving. We describe clinicopathological and immunohistochemical features of five myoepithelial carcinomas, including molecular cytogenetic results in one case. There were five male patients within age-range of 8-40 years (median = 26). Four tumours occurred in the long bones, including two tumours, each, in the femur and fibula, respectively, while a single tumour occurred in the proximal phalanges. Tumour size (n = 3 cases) varied from 5.6 to 8.6 cm. On radiological imaging, most tumours appeared as expansile, lytic and destructive lesions. Two tumours appeared as sclerotic lesions. Two cases were referred with diagnoses of chondrosarcomas and a single case was referred with two different diagnoses, including an adamantinoma and an osteosarcoma. Histopathological examination in all these cases showed multinodular tumours comprising mostly polygonal cells, exhibiting moderate nuclear atypia and interspersed mitotic figures within a stroma containing variable amount of myxoid, chondroid, hyalinised and osteoid-like material. Three tumours revealed prominent squamous differentiation. By immunohistochemistry, tumour cells were positive for EMA (5/5), pan CK (AE1/AE3) (3/3), CK5/6 (4/4), CK MNF116 (1/1), S100 protein (5/5) and GFAP (3/5). The first tumour revealed EWSR1 rearrangement. The first patient, 10 months after tumour resection and a simultaneous lung metastatectomy, is free-of-disease (FOD). The second patient, 11 months after tumour resection is FOD. The third and fourth patients underwent wide resections and are on follow-up. The fifth patient underwent resections, including a lung metastatectomy. Primary intraosseous myoepithelial carcinomas are rare and mimic conventional primary bone tumours. Some primary intraosseous myoepithelial carcinomas display EWSR1 rearrangement. Squamous differentiation may be considered as an addition to their evolving histopathological spectrum. Immunohistochemical stains constitute as a necessary tool for arriving at the correct diagnosis in such cases, which has treatment implications. Surgical resection remains the treatment mainstay.

Adamantinoma-like Ewing family tumors of the head and neck: a pitfall in the differential diagnosis of basaloid and myoepithelial carcinomas.

Ewing sarcoma family tumors (EFTs) of the head and neck are rare and may be difficult to diagnose, as they display significant histologic overlap with other more common undifferentiated small blue round cell malignancies. Occasionally, EFTs may exhibit overt epithelial differentiation in the form of diffuse cytokeratin immunoexpression or squamous pearls, resembling the so-called adamantinoma-like EFTs and being challenging to distinguish from bona fide carcinomas. Furthermore, the presence of EWSR1 gene rearrangement correlated with strong keratin expression may suggest a myoepithelial carcinoma. Herein, we analyze a series of 7 adamantinoma-like EFTs of the head and neck, most of them being initially misdiagnosed as carcinomas because of their anatomic location and strong cytokeratin immunoexpression, and subsequently reclassified as EFT by molecular techniques. The tumors arose in the sinonasal tract (n=2), parotid gland (n=2), thyroid gland (n=2), and orbit (n=1), in patients ranging in age from 7 to 56 years (mean, 31 y). Microscopically, they departed from the typical EFT morphology by growing as nests with peripheral nuclear palisading and prominent interlobular fibrosis, imparting a distinctly basaloid appearance. Moreover, 2 cases exhibited overt keratinization in the form of squamous pearls, and 1 sinonasal tumor demonstrated areas of intraepithelial growth. All cases were positive for CD99, pancytokeratin, and p40. A subset of cases showed synaptophysin, S100 protein, and/or p16 reactivity, further confounding the diagnosis. Fluorescence in situ hybridization assays showed EWSR1 and FLI1 rearrangements in all cases. Our results reinforce that a subset of head and neck EFTs may show strong cytokeratin expression or focal keratinization, and are therefore histologically indistinguishable from more common true epithelial neoplasms. Thus, CD99 should be included in the immunopanel of a round cell malignancy regardless of strong cytokeratin expression or anatomic location, and a strong and diffuse CD99 positivity should prompt molecular testing for the presence of EWSR1 gene rearrangements.

Activating GNAS mutations in parosteal osteosarcoma.

Parosteal osteosarcoma is a surface-based osteosarcoma that often exhibits deceptively bland cytologic features, hindering diagnosis in small biopsies or when correlative radiologic imaging is not readily available. A number of benign and malignant fibro-osseous lesions, including fibrous dysplasia (FD) and low-grade central osteosarcoma, fall within the morphologic differential diagnosis of parosteal osteosarcoma. Somatic mutations in GNAS, encoding the α-subunit of the heterotrimeric G protein complex (Gsα), occur in FD and McCune-Albright syndrome but have not been reported in parosteal osteosarcoma. We evaluated GNAS mutational status in parosteal osteosarcoma and several of its histologic mimics to determine its utility in differentiating these entities. Eleven of 14 (79%) FD cases had GNAS mutations within codon 201 (5 R201C and 6 R201H mutations). GNAS mutations were not detected in any cases of adamantinoma or osteofibrous dysplasia. Direct sequencing of 9 parosteal osteosarcomas, including 3 of low grade and 6 with dedifferentiation, revealed activating GNAS mutations in 5 cases (55%), distributed as 4 R201C-mutated tumors and 1 tumor with an R201H mutation. GNAS codon 227 mutations were not detected in any of the cases. There was no association between GNAS mutational status and patient demographics, histologic dedifferentiation, or clinical outcome. To our knowledge, we report the first series of parosteal osteosarcomas harboring activating GNAS mutations. Our data suggest that GNAS mutational status may have limited utility as an ancillary technique in differentiating benign and malignant fibro-osseous lesions of the bone.

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.

Osteofibrous dysplasia and adamantinoma in children and adolescents: a clinicopathologic reappraisal.

Osteofibrous dysplasia (OFD) and adamantinoma are rare and most commonly arise in the tibia of young individuals. Although OFD has typical histopathologic features, areas resembling OFD have often been noted at the periphery of otherwise classic adamantinomas, and some have suggested that OFD may be either a precursor to or a regressive phase of adamantinoma. The so-called OFD-like adamantinoma encompasses some features of both OFD and adamantinoma. We studied the clinical, imaging, histopathologic, immunohistochemical, ultrastructural, and molecular features of 16 OFD and 8 adamantinomas (5 OFD-like and 3 classic) in an attempt to further define their morphology, clinical course, and relationship. Patients with OFD were generally younger than those with adamantinoma. Osteoblastic and osteoclastic activity was more prominent in OFD than in OFD-like adamantinoma. In addition to the inconspicuous small clusters of epithelial cells in OFD-like adamantinoma, isolated keratin-positive cells with a unique ultrastructural hybrid fibroblastic-epithelial phenotype were found in the stroma of all OFD and OFD-like adamantinomas. Fluorescence in situ hybridization analysis revealed trisomies 7, 8, and/or 12 in the spindle cell stroma of OFD, OFD-like, and classic adamantinoma, supporting a neoplastic origin of OFD and a common histogenesis for all 3 lesions. Trisomies were not observed in osteoblasts or osteoclasts suggesting that the osseous component is reactive and non-neoplastic. Of the 11 OFD patients with follow-up (median, 4.5 y), all 3 who underwent incisional biopsy had persistent, nonprogressive disease and 2 of 8 who underwent curettage or wide excision had recurrence; none developed adamantinoma. All 6 adamantinoma patients with follow-up (3 classic and 3 OFD-like) were treated with wide excision. One with classic adamantinoma died of pulmonary metastases 9 years after presentation; the other 5 were free of disease with a median follow-up of 12 years. None of the classic adamantinomas evolved into OFD-like adamantinoma or OFD. Although the histopathology, immunohistochemistry, ultrastructure, and cytogenetics indicate that these lesions are closely related, our data and the literature suggest that only classic adamantinoma has malignant potential. OFD, OFD-like adamantinoma, and classic adamantinoma appear to show a progressive complexity of cytogenetic aberrations, perhaps indicative of a multistep neoplastic transformation.

Adamantinoma-like Ewing's sarcoma with EWS-FLI1 fusion gene: a case report.

Recent studies have advocated the genotypic and phenotypic delineation of a novel Ewing's sarcoma histologic variant showing epithelial features defined as "adamantinoma-like Ewing's sarcoma". We described an 18-year-old girl with a primary small round-cell sarcoma of the right tibia showing polyphenotypic differentiation with epithelioid features. The neoplastic cells had mainly round or oval nuclei with fine chromatin with a portion of epithelial arrangements. The immunohistochemical analysis showed the epithelial markers of cytokeratin 5/6/18, AE1/AE3, and cytokeratin high molecular weight were stained especially in the foci with epithelioid features, as well as MIC2, S100, and NSE. The diagnosis of the lesion was confirmed as Ewing's sarcoma by the presence of the EWS-FLI1 fusion transcript, and could be defined as the so-called "adamantinoma-like Ewing's sarcoma". After wide excision and high-dose chemotherapy with peripheral blood stem cell transfusion, the patient has been well and continuously event-free for 3 years since the initial diagnosis.