Absence of EWS/FLI1 fusion in olfactory neuroblastomas indicates these tumors do not belong to the Ewing's sarcoma family

The balanced reciprocal translocation t(11;22)(q24;q12) is a specific molecular marker for the Ewing's sarcoma family of tumors (ESFT). Based on the detection of this translocation in some olfactory neuroblastomas (ONBs), it has been proposed that ONBs also belong to the ESFT. Others have challenged this hypothesis; however, the rarity of ONBs, as well as the lack of molecular techniques that work reliably on formalin fixed tissue, have precluded the molecular analysis of a significant number of cases. We evaluated the immunophenotypic and molecular features of 17 paranasal small round neuroectodermal tumors using routinely fixed tissue. Probes localizing to 22q12 (EWS) and 11q24 (FLI-1) were used in a standard 2-color fluorescence in situ hybridization (FISH) assay to evaluate EWS/FLI1 fusion on the der(22). Sixteen tumors were mic-2 negative, whereas 1 tumor was mic-2 positive, compatible with ONB and ESFT, respectively. Thirteen of 15 ONBs could be evaluated by FISH, and all 13 were negative for the EWS/FLI1 fusion. Distinct fusion signals were identified in the single paranasal ESFT. Our findings indicate that ONBs lack the EWS/FLI1 fusion and are unrelated to the ESFT; however, true ESFTs may rarely occur as primary sinonasal tumors.     

Solid pseudopapillary neoplasms of the pancreas are associated with FLI-1 expression, but not with EWS/FLI-1 translocation.

Solid pseudopapillary neoplasms of the pancreas are rare pancreatic tumors with mostly benign behavior, affecting almost exclusively women. Their histogenetic origin is still unsolved, but a recently reported EWS/FLI-1 translocation t(11;22)(q24;q21) and the consistent expression of CD56 and the progesterone receptor, both genes located on the long arm of chromosome 11, point to chromosome 11q as a potential locus of gene aberration in solid pseudopapillary neoplasms. To further elucidate this issue, we studied 30 cases of solid pseudopapillary neoplasms by comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH) and immunohistochemistry. Immunohistochemically, 38% showed nuclear expression of FLI-1 and all cases revealed positivity for CD56 and the progesterone receptor, whereas no solid pseudopapillary neoplasm expressed CD34. No translocation of the EWS gene was found by FISH and no gross chromosomal gain or loss was detected by CGH. It is concluded that FLI-1 expression in solid pseudopapillary neoplasms is not associated with an EWS/FLI-1 translocation. In addition, there are no chromosomal gains or losses, especially on chromosome 11, where the FLI-1 gene is located adjacent to the gene for CD56 (NCAM). These data add another feature to the complex phenotypic appearance of solid pseudopapillary neoplasms.     

Ewing sarcoma/peripheral primitive neuroectodermal tumor: adult abdominal tumors with an Ewing sarcoma gene rearrangement demonstrated by fluorescence in situ hybridization in paraffin sections.

The differential diagnosis of small round cell tumors is exhaustive and requires ancillary studies. Relatively recently, fluorescence in situ hybridization (FISH) using probes for specific gene rearrangements has gained wide acceptance. This technique is particularly useful in the differential diagnosis of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) and desmoplastic small round-cell tumor (DSRCT). In ES/PNET, the EWS gene is juxtaposed to the FLI-1 gene in 85% of cases and to the ERG gene in another 7% of cases; the EWS gene is juxtaposed to the WTI gene in DSRCT. Documentation of the EWS gene rearrangements in EWS/PNET has previously been demonstrated in frozen tissue. We report 2 unusual cases of EWS/PNET diagnosed in abdominal tumors in adults. Although the immunohistochemical results supported a diagnosis of ES/PNET, 1 case morphologically resembled DSRCT. The diagnosis in these 2 cases was confirmed by the FISH demonstration of EWS/FLI-1 gene fusion in paraffin-embedded tissue. Thus, the usefulness of FISH demonstration of an EWS gene rearrangement with these specific probes in such unusual cases is supported and is demonstrated in paraffin-embedded tissue.     

A practical approach to the clinical diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue

BACKGROUND: Over 90% of Ewing's sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. AIMS: To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS. METHODS: Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. RESULTS: One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls. CONCLUSIONS: Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.     

荧光原位杂交与逆转录聚合酶链反应在尤文肉瘤/原始神经外胚层肿瘤分子诊断中的应用

目的探讨运用荧光原位杂交（FISH）与逆转录聚合酶链反应（RT-PCR）检测尤文肉瘤/原始神经外胚层肿瘤（ES/PNET）石蜡包埋组织特异性染色体易位的临床应用价值.方法收集ES/PNET 10例,分离石蜡包埋组织中的肿瘤细胞,利用Vysis公司的EWSR1双色易位分离探针,间期核FISH检测EWS基因的易位.运用RT-PCR检测t（11;22）（q24;q12）和t（21;22）（q22;q12）形成的融合转录子EWS-FLI1和EWS-ERG.结果10例ES/PNET中FISH观察9例有EWS基因的易位.RT-PCR 8例检测出EWS-FLI1的表达,没有检测出EWS-ERG的表达.结论利用FISH与RT-PCR检测ES/PNET石蜡包埋组织特异性染色体易位可作为可靠的分子诊断指标;两者相比,FISH敏感性和稳定性要优于RT-PCR.     

Diagnostic utility of FLI-1 monoclonal antibody and dual-colour, break-apart probe fluorescence in situ (FISH) analysis in Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET). A comparative study with CD99 and FLI-1 polyclonal antibodies

AIMS: To compare the sensitivity and specificity of the recently commercially available FLI-1 monoclonal (FLI-1m) antibody with the currently used antibodies [CD99 and FLI-1 polyclonal (FLI-1p)] in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET) and to determine the diagnostic value of the EWSR1 (22q12) dual-colour, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique. MATERIALS AND METHODS: Forty-three cases of well-documented EWS/PNET and 15 non-EWS/PNET cases were retrieved from the archival files. Immunohistochemistry (IHC) for FLI-1p, FLI-1m and FISH analysis was performed. RESULTS: The most sensitive and specific test panel for the diagnosis of EWS/PNET is the combination of CD99 and FLI-1p. FISH had a very high specificity (100%) but only a moderate sensitivity (50%). CONCLUSION: The combination of CD99 and FLI-1p is the method of choice for the diagnosis of EWS/PNET. EWRS1 (22q12) dual-colour, break-apart rearrangement probe FISH should be used as a confirmatory test in addition to CD99 and FLI1-p due to its high specificity.     

Keratin-positive Ewing's sarcoma: an ultrastructural study of 12 cases

Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET) is an aggressive neoplasm of bone and soft tissue. Histologically, it is characterized by the presence of small round blue cells, which usually express MIC-2 and FLI-1 immunohistochemically. The most specific feature for diagnosis, however, is cytogenetic or molecular evidence of a consistent abnormality, the t(11;22)(q24;q12), or variants thereof. The immunohistochemical expression of keratins in a significant proportion of these cases has been highlighted in several recent studies. The ultrastructural features of these keratin-positive tumors have not, however, been characterized in detail. In this study we analyzed the ultrastructural features of 12 well-documented EWS/PNETs that stained strongly for pankeratin by immunohistochemistry. Ultrastructurally, the tumor cells contained a few organelles, which included a small number of mitochondria, poorly developed Golgi complexes, free ribosomes, and inconspicuous rough-endoplasmic reticulum. Rudimentary cell junctions were seen in 2 tumors while prominent junctions were observed in the remaining 10. Five tumors contained intracytoplasmic filaments, and definite tonofibrils were identified in 2. Well-developed basal lamina around tumor cells were also demonstrated in 2 tumors. Follow-up information was available for all cases. Seven patients died of disease, 2 are alive with disease, and 3 have no current evidence of disease. The cohort includes 5 patients with a type-1 translocation, which has been associated with a better prognosis in some studies; 4 of these patients have died of their disease, and 1 is alive with recurrent disease. This study shows that keratin-positive EWS/PNETs have evidence of epithelial differentiation ultrastructurally, and may possibly represent a more aggressive subset of the EWS/PNET group of tumors.     

Fluorescence in situ hybridization analysis of complex translocations in two newly diagnosed Philadelphia chromosome-positive chronic myelogenous leukemia patients.

Two different complex translocations from newly diagnosed cases of Philadelphia chromosome-positive chronic myelogenous leukemia (CML) were characterized by G-banding and fluorescence in situ hybridization (FISH) analysis. In one case, a unique balanced t(9;22;9;11) (q34;q11;p22;q23) was identified by G-banding, and confirmed by FISH using MBCR/ABL and painting probes. In the second case, an apparently balanced t(19;22) was identified by G-banding analysis. FISH using MBCR/ABL probe detected the fusion gene on the derivative chromosome 22, indicating the involvement of chromosome 9. Further FISH analysis with selected painting probes showed that the t(19;22) was a result of a complex translocation involving chromosomes 9, 19, 21, and 22.