Adamantinoma-like Ewing Sarcoma of the thyroid gland: Cytomorphologic, phenotypic and molecular features.

Adamantinoma-like Ewing Sarcoma (ALES) is a rare subtype of Ewing sarcoma family of tumors (EFTs) which are defined by their EWSR1 gene rearrangements. We present a case of a 15-year old female with a swelling in her anterior neck of 4 months duration which had recently begun to rapidly grow in size. Fine needle aspiration showed a small blue round cell tumor with immunoreactivity for cytokeratin, CD99 and FLI1. Material for molecular testing was available on the resection specimen. Demonstration of t(11;22) (EWS-FLI1) was helpful in establishing the diagnosis.

[Histopathology and molecular profile of a case of prostatic Ewing sarcoma]. / Histopatología y perfil molecular de un caso de sarcoma de Ewing prostático.

We report a case of Ewing sarcoma localized in the prostate gland of a 33-year-old patient without bone or soft tissue involvement. Evidence of EWS and FLI1 gene translocation was detected by fluorescence in situ hybridization (FISH). This is an unusual case with an interesting clinical presentation; indeed, only a few cases have been reported to date and not all have the supporting biological studies now considered essential for the diagnosis.

Ewing sarcoma.

Ewing sarcoma is the second most frequent bone tumour of childhood and adolescence that can also arise in soft tissue. Ewing sarcoma is a highly aggressive cancer, with a survival of 70-80% for patients with standard-risk and localized disease and ~30% for those with metastatic disease. Treatment comprises local surgery, radiotherapy and polychemotherapy, which are associated with acute and chronic adverse effects that may compromise quality of life in survivors. Histologically, Ewing sarcomas are composed of small round cells expressing high levels of CD99. Genetically, they are characterized by balanced chromosomal translocations in which a member of the FET gene family is fused with an ETS transcription factor, with the most common fusion being EWSR1-FLI1 (85% of cases). Ewing sarcoma breakpoint region 1 protein (EWSR1)-Friend leukaemia integration 1 transcription factor (FLI1) is a tumour-specific chimeric transcription factor (EWSR1-FLI1) with neomorphic effects that massively rewires the transcriptome. Additionally, EWSR1-FLI1 reprogrammes the epigenome by inducing de novo enhancers at GGAA microsatellites and by altering the state of gene regulatory elements, creating a unique epigenetic signature. Additional mutations at diagnosis are rare and mainly involve STAG2, TP53 and CDKN2A deletions. Emerging studies on the molecular mechanisms of Ewing sarcoma hold promise for improvements in early detection, disease monitoring, lower treatment-related toxicity, overall survival and quality of life.

A SMARCB1-deficient vulvar neoplasm with prominent myxoid stroma: report of a case showing ERG and FLI1 expression.

In the vulvar region, epithelioid sarcoma (ES) is the most frequent SMARCB1-deficient neoplasm, followed by myoepithelial carcinoma (MC). Previous studies have demonstrated that some SMARCB1-deficient vulvar neoplasms cannot be classified as either ES or MC. Herein, we report of a 42-year-old woman with a SMARCB1-deficient neoplasm with prominent myxoid stroma in the vulva. It contained both epithelioid and spindled tumor cells, both of which showed vimentin and EMA expression. Although other markers useful for the differential diagnosis among SMARCB1-deficient tumors were negative, this tumor displayed characteristic expression of ERG and FLI1. As there are no reliable data regarding expression of ERG and FLI1 in MC, which are demonstrated to be often expressed in ES, further classification of cases such as the one reported here requires reliable data regarding their expression status in MC.

NLS-tagging: an alternative strategy to tag nuclear proteins.

The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the 'tag' close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis.

Immunolabeling for p16, WT1, and Fli-1 in the assignment of growth phase for cutaneous melanomas.

Distinction between radial growth phase (RGP) and vertical growth phase (VGP) in cutaneous melanomas is prognostically significant. Despite established morphological criteria, molecular markers to separate RGP and VGP have not been well established. The goal of this study was to investigate associations of p16, WT1, and Fli-1 with RGP-to-VGP progression, by immunohistochemistry. The p16 is a tumor suppressor, whereas WT1 and Fli-1 are transcriptional activators. The authors hypothesized that entry into VGP would be associated with decreased p16 and increased WT1 and Fli-1. Paraffin sections from 18 RGP and 15 VGP melanomas were immunostained with well-characterized antibodies to p16, WT1, and Fli-1. Melanoma growth phases were determined using precodified morphological attributes. In RGP melanomas, p16 was expressed in 15 of 18 (83%), WT1 in 17 of 17 (100%), and Fli-1 at least focally in 6 of 18 (33%). The deep dermal component of VGP melanomas stained positively for Fli-1 in 9 of 14 (64%), strongly for WT1 in 10 of 14 (71%), and strongly for p16 in only 2 of 15 (13%). Observed patterns of WT1 immunopositivity did not support the authors' hypothesis; it is not likely to be a good indicator of VGP. On the other hand, Fli-1 staining trended toward more positive deep tumor compartment staining and p16 to weaker staining in the deep compartment. At present, application of histological criteria remains the best method for assignment of growth phase in melanomas; however, p16 and possibly Fli-1 immunostains may serve as useful adjuncts in morphologically indeterminate cases.

ERG and FLI1 protein expression in epithelioid sarcoma.

Epithelioid sarcoma is a rare, aggressive keratin-positive sarcoma that co-expresses CD34 in 50% of cases and may mimic an angiosarcoma. Recently, we have observed one case of epithelioid sarcoma that labeled for ERG, an ETS family regulatory transcription factor, which is considered to be a reliable marker for vascular differentiation. We investigated the prevalence of nuclear expression of ERG and FLI1, a homologous transcription factor, in these tumors. A formalin-fixed paraffin-embedded tissue microarray of 37 epithelioid sarcomas was examined. Immunohistochemistry was performed using anti-ERG monoclonal antibody to the N-terminus, anti-ERG monoclonal antibody to the C-terminus and anti-FLI1 monoclonal antibody. Comparison was made with CD34, CD31, and D2-40 labeling. The extent of immunoreactivity was graded according to the percentage of positive tumor cell nuclei (0: no staining; 1+: <5%; 2+: 5-25%; 3+: 26-50%; 4+: 51-75%; and 5+: 76-100%), and the intensity of staining was graded as weak, moderate, or strong. Nuclear staining for the N-terminus of ERG was seen in 19 out of 28 cases: 10 with diffuse(4 to 5+) strong/moderate labeling; 1 with 2+ moderate labeling and 8 with weak labeling (1 to 4+, 2 each). Focal staining for the C-terminus of ERG was seen in only 1 out of 29 cases (2+ moderate). FLI1 labeling was seen in nearly all (28 out of 30) cases: 16 with diffuse (5+) predominantly moderate labeling, and 8 cases with diffuse(5+) weak labeling. The remainder had variable moderate (1 to 3+) or weak (1 to 4+) FLI1 staining. CD34 was positive in 22 out of 30 cases and D2-40 was found to be positive in 22 out of 31 cases. All cases were negative for CD31 (0 out of 30). Epithelioid sarcoma can label with antibodies to the N-terminus of ERG, FLI1, and D2-40, which may cause diagnostic confusion for a vascular tumor. A panel of other antibodies including SMARCB1 and CD31 should be used in evaluating these tumors. ERG antibody selection is also critical, as those directed against the C-terminus are less likely to label epithelioid sarcoma.

Diagnostic utility of Fli-1 and D2-40 in distinguishing atypical fibroxanthoma from angiosarcoma.

Although in most cases one can easily distinguish between atypical fibroxanthomas and angiosarcomas, hemorrhagic atypical fibroxanthomas can pose a diagnostic problem. In rare cases, the large atypical cells of atypical fibroxanthoma can stain with CD31, leading to the erroneous diagnosis of angiosarcoma. We elected to further study this conundrum with 2 additional markers of lymphatic and vascular elements, namely D2-40 (podoplanin) and Fli-1, respectively. We studied 26 cases of atypical fibroxanthoma and 20 cases of angiosarcoma with Fli-1 and D2-40. We found that both Fli-1 and D2-40 stained a majority of cases of angiosarcoma (16/20 and 12/20, respectively), although only staining a minority of cases of atypical fibroxanthoma (8/26 for both). In addition, D2-40 staining of atypical fibroxanthoma was usually weak when positive, whereas Fli-1 staining of angiosarcomas was mostly strong and nuclear. Thus, both D2-40 and Fli-1 seem to be useful in distinguishing between atypical fibroxanthomas and angiosarcomas.

Utility of immunohistochemical staining with FLI1, D2-40, CD31, and CD34 in the diagnosis of acquired immunodeficiency syndrome-related and non-acquired immunodeficiency syndrome-related Kaposi sarcoma.

CONTEXT: Kaposi sarcoma (KS) is a vascular tumor frequently associated with advanced human immunodeficiency virus infection, advanced age, or iatrogenic immunosuppression. Immunohistochemistry for CD31 and CD34, and more recently for FLI1 and D2-40, has been used as ancillary diagnostic tests for KS, despite little information regarding the sensitivities and differential staining patterns of the latter 2 markers in the major clinical subtypes and histologic stages of KS. OBJECTIVE: This retrospective study aims to assess the prevalence of the vascular markers D2-40 and FLI1 in the main clinical subgroups and tumor stages of KS. DESIGN: Twenty-four cases of KS (12 acquired immunodeficiency syndrome [AIDS]-related cases and 12 non-AIDS-related cases; 11 nodular-stage and 13 patch/plaque-stage KS) were stained for CD34, CD31, D2-40, and FLI1 by immunohistochemistry. The distribution of immunoreactivity was compared between the clinical subtypes and tumor stages of KS using the Mann-Whitney test. RESULTS: CD31, CD34, D2-40, and FLI1 strongly and diffusely stained tumor cells in 75%, 92%, 67%, and 92% of AIDS-related cases and 58%, 92%, 67%, and 75% of non-AIDS-related cases, respectively. Differences in the proportions of positive cases between AIDS-related and non-AIDS-related cases did not reach statistical significance. No significant staining differences were observed between nodular- and patch/plaque-stage KS either. CONCLUSIONS: There are no differences in the distribution of immunohistochemical reactivity for CD31, CD34, D2-40, or FLI1 between AIDS-related and non-AIDS-related KS or between nodular- and patch/plaque-stage KS. All of the markers studied demonstrated high sensitivity in both clinical settings and both stages of tumor progression.

[An unusual adrenal tumor: Ewing tumor]. / Une tumeur surrénalienne inhabituelle : tumeur d'Ewing.

We report the case of a voluminous tumor of the adrenal diagnosed in a young pregnant woman at 26(th) week of amenorrhea. Morphologically, a soft white tumor with haemorragic areas was observed, made of sheets of monomorphous, medium sized, spindle-shaped to polygonal, with high mitotic activity. Tumorous cells expressed cytokeratins AE1/AE3, EMA, and CD99 (expression of vimentin is not relevant). Contemplated diagnoses included poorly differentiated synovialosarcoma, sarcomatoid carcinoma and Ewing tumor. Thanks to molecular biology, showing the specific transcript of Ewing/peripheral primitive neuroectodermal tumor (pPNET) EWS/FLI1, the diagnosis of this atypical tumor in an unusual location was performed. Indeed, 75% of Ewing tumors involve bones (especially, the diaphysis of long bones) and 20 to 25% soft tissues. Primitive visceral involvement is rare; less than 10 cases of adrenal involvement have been reported. The hypothesis that Ewing cell's origin is a mesenchymal stem cell, which may derive from neural crest cell, could explain the uncommon adrenal involvement. Diagnosis of Ewing tumor is based on pathologic and molecular findings, especially in atypical cases.

Unusual myogenic and melanocytic differentiation of soft tissue pPNETs: an immunohistochemical and molecular study of 3 cases.

All of the members of the peripheral primitive neuroectodermal tumor family (Ewing sarcomas, neuroectodermal tumors of bone, peripheral neuroepitheliomas, and Askin tumors) have similar morphologic and immunophenotypical features (ie, the proliferation of small and medium-sized round cells in a fibrous background showing strong and diffuse immunohistochemical positivity for CD99), and the common cytogenetic abnormality of a nonrandom translocation involving the EWS gene and one of several members of the erythroblastosis virus transforming sequence family of transcription factors. The combination of clinical information and morphologic/immunophenotypical characteristics is usually sufficient for a correct diagnosis, but there are rare cases in which an unusual predominant or multidirectional immunophenotypical differentiation makes diagnosis a challenge and requires the use of molecular cytogenetic or molecular techniques. We describe 3 such cases in which we employed fluorescence in-situ hybridization analysis to detect translocation involving the EWS gene and reverse transcription polymerase chain reaction followed by sequencing to detect the fusion transcript EWS-FLI1.

Comparative analysis of routine histology, immunohistochemistry, reverse transcriptase polymerase chain reaction, and fluorescence in situ hybridization in diagnosis of Ewing family of tumors.

CONTEXT: The Ewing family of tumors are often difficult to distinguish from other malignant small round cell tumors, but more than 90% have EWS-FLI1 chimeric transcript, which acts as a potential molecular diagnostic marker. OBJECTIVE: To do a comparative analysis of 32 cases with EWS-FLI1: Ewing family of tumors (n = 30), desmoplastic small round cell tumor (n = 1), and undifferentiated sarcoma (n = 1). DESIGN: The initial diagnosis was made on core biopsy (n = 22) and open biopsy (n = 4) specimens by using morphology and immunohistochemistry and on fine-needle aspiration cytology ([FNAC], n = 6) specimens. EWS-FLI1 was detected by reverse transcriptase polymerase chain reaction on all 32 fresh FNAC samples and by fluorescence in situ hybridization on 16 paraffin blocks. RESULTS: The 19 male and 13 female patients had bone (n = 19) or soft tissue (n = 13) tumors. Histologic groups were typical Ewing sarcoma (n = 15), atypical Ewing sarcoma (n = 4), Askin Rosai tumors (n = 5), desmoplastic small round cell tumor (n = 1), undifferentiated sarcoma (n = 1), and cases diagnosed as malignant small round cell tumors on FNAC (n = 6). All tumors except desmoplastic small round cell tumor and undifferentiated sarcoma were CD99 positive. EWS-FLI1 by reverse transcriptase polymerase chain reaction was noted in 15 cases of typical Ewing sarcoma, 4 cases of atypical Ewing sarcoma, 5 cases of Askin Rosai tumor, and no cases of desmoplastic small round cell tumor or undifferentiated sarcoma. With use of fluorescence in situ hybridization, EWS break was detected in 10 of 11 paraffin blocks used and was negative in desmoplastic small round cell tumor. CONCLUSIONS: The excellent correlation of routine histologic findings in Ewing family of tumors with results on immunohistochemistry and fluorescence in situ hybridization on archival material and reverse transcriptase polymerase chain reaction on fresh FNAC specimens underscores that the traditional observation on routine histologic examination is a time-tested tool. The diagnosis of Ewing family of tumors can be validated on archival material or fresh biopsy samples, including those obtained by FNAC.

Ewing sarcoma family of tumors in unusual sites: confirmation by rt-PCR.

Ewing sarcoma family tumors originating in the palate or adrenal gland are extremely rare and may cause difficulty in diagnosis. More common tumors primary to these sites need to be excluded before one arrives at the correct diagnosis. We have recently diagnosed 2 such cases. The 1st case was that of a 24-year-old woman who presented with a swelling in the right side of the hard palate. The 2nd case was diagnosed in a 28-year-old woman who presented with a mass in the right adrenal gland. In both cases, the diagnosis of Ewing sarcoma family of tumors was confirmed by immunohistochemical studies and reverse transcriptase-polymerase chain reaction (RT-PCR). The hard palate case is the 1st and the adrenal gland the 3rd case of Ewing sarcoma family of tumors arising in these sites, in which the diagnosis was confirmed by RT-PCR and/or cytogenetics. Accurate diagnosis of Ewing sarcoma family of tumors is crucial for the management of patients, and when found in such rare locations, diagnosis should be supported by immunohistochemical and/or molecular genetic studies.

Primitive neuroectodermal tumor of the uterus: a case report and review of literature.

A case of primitive neuroectodermal tumor arising in the uterine corpus of a 43-year-old woman is presented. The tumor mass was 13.3 cm and extended to the uterine serosa, endocervical stroma, and left adnexa. Histologically, the tumor was composed of small blue cells with scant cytoplasm, indistinct cell borders, hyperchromatic round nuclei, and inconspicuous nucleoli focally forming pseudorosettes, suggestive of neuroectodermal origin. The tumor cells displayed strong immunoreactivity for CD99 and FLI1. Cytogenetic fluorescence in situ hybridization study revealed presence of an EWS-FLI1 fusion gene. To the best of our knowledge, this is the first case of primitive neuroectodermal tumor of the uterus with diagnosis confirmed by FLI1 immunohistochemical labeling and demonstration of t (11; 22) by fluorescence in situ hybridization.

Cluster analysis of immunohistochemical profiles in synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma.

As a result of overlapping morphologic and immunohistochemical features, it can be difficult to distinguish synovial sarcoma, malignant peripheral nerve sheath tumor, and Ewing sarcoma/primitive neuroectodermal tumor in core biopsies. To analyze and compare immunohistochemical profiles, we stained tissue microarrays of 23 synovial sarcomas, 23 malignant peripheral nerve sheath tumors, and 27 Ewing sarcomas with 22 antibodies potentially useful in the differential diagnosis, and analyzed the data with cluster analysis. Stain intensity was scored as none, weak, or strong. For CD99, tumors with membranous accentuation were independently categorized. Cluster analysis sorted five groups, with like tumors clustering together. Synovial sarcoma clustered into two groups: one cytokeratin and EMA positive (n = 11), the other mostly cytokeratin negative, EMA positive, bcl-2 positive and mostly CD56 positive (n = 9). Malignant peripheral nerve sheath tumor clustered into two groups: one S100 positive, with nestin and NGFR positivity in most (n = 10), the other mostly S100 negative, and variably but mostly weakly positive for nestin and NGFR (n = 11). Ewing sarcomas clustered into a single group driven by membranous CD99 staining. Thirteen cases failed to cluster (outliers), while three Ewing sarcomas clustered into groups of other tumor types. Paired antibodies for each tumor type determined by visual assessment of cluster analysis data and statistical calculations of specificity, sensitivity, and predictive values showed that EMA/CK7 for synovial sarcoma, nestin/S100 for malignant peripheral nerve sheath tumor, and membranous CD99/Fli-1 for Ewing sarcoma yielded high specificity and positive predictive values. Cluster analysis also highlighted aberrant staining reactions and diagnostic pitfalls in these tumors. Hierarchical cluster analysis is an effective method for analyzing high-volume immunohistochemical data.