Cytogenetic and molecular cytogenetic studies of a variant of t(21;22), ins(22;21)(q12;q21q22), with a deletion of the 3' EWSR1 gene in a patient with Ewing sarcoma

Ewing sarcoma is the second most common malignant bone tumor in children and young adults. Cytogenetic analysis to identify a common t(11;22)(q23;q12) or less frequently a t(21;22)(q22;q12) or t(7;22)(p22;q12) plays an important role in the confirmation of the clinical diagnosis. We report a case of a 10-year-old female who had extraskeletal Ewing sarcoma. Conventional cytogenetic analysis revealed that 11 out of 20 cells had a derivative chromosome 22, possibly due to an insertion of the long arm of the 21q21 approximately q22. This finding was confirmed by fluorescence in situ hybridization (FISH) utilizing whole chromosome paint probes specific for chromosomes 21 and 22. Hybridization utilizing LSI EWSR1, dual-color break-apart rearrangement probe unexpectedly revealed that the 3' EWSR1 gene was lost on the derivative chromosome 22. This finding suggests that the insertion of chromosome 21 is another mechanism that could lead to EWS-ERG gene fusion. To our knowledge, this is the first case report of an insertion of a segment of 21q21 approximately q22 into the long arm of 21q12 with a loss of a DNA segment around the breakpoint on the derivative chromosome 22 in Ewing sarcoma.     

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.     

Molecular cytogenetic analysis of a complex t(10;22;11) translocation in Ewing's sarcoma.

Fluorescence in situ hybridization (FISH) using chromosome-specific plasmid libraries and chromosome region-specific DNA markers allowed the characterization of a t(10;22;11) (p11.2;q12;q24) in a Ewing's sarcoma (ES). This study illustrates the usefulness of molecular cytogenetic analysis of ES, especially for determining the localization of the translocated 11q24-25 segment in complex or variant translocations.     

Chromosomes in Ewing's sarcoma. II. Nonrandom additional changes, trisomy 8 and der(16)t(1;16)

Chromosomal data from 82 informative, unrelated Ewing's sarcoma (ES) specimens (including 20 personal specimens) were reviewed for secondary changes additional to the t(11;22)(q24;q12). Additional numerical and/or structural changes were found in 75 specimens. Trisomy 8 was observed consistently in half of the 43 cases selected for analysis of numerical changes. A nonrandom der(16) was observed as a result of an unbalanced t(1;16) in 18% of the 82 analyzed for structural changes. Consistent involvement of chromosome #16 in rearrangements with chromosome #1 may be an additional chromosome change specifically associated with ES.     

Chromosomes in Ewing's sarcoma. I. An evaluation of 85 cases of remarkable consistency of t(11;22)(q24;q12)

Since our initial reports on chromosomal studies in eight Ewing's sarcomas (ES), we have carried out similar investigations on 23 additional ES specimens following short-term culture of tumor cells (16 cases), and established in vitro cell lines (three cases) and on xenografted tumors in nude mice (four cases). We demonstrated the presence of the reciprocal t(11;22)(q24;q12) in every case except one that exhibited a complex t(11;22;14)(q24;q12;q11). On the basis of results from these additional 23 cases, we confirm the consistency of the t(11;22)(q24;q12) in ES. Moreover, we reviewed 54 ES cases reported by other investigators; when added to our 31 cases, this brings the total number to 85 unrelated cases of ES available for an evaluation of the frequency of involvement of bands 11q24 and 22q12 in translocations in ES. The standard t(11;22)(q24;q12) proved to be a remarkably consistent event, present in 83% of the cases. Five percent of the cases exhibited complex translocations involving a third chromosome in addition to chromosomes #11 and #22. In 4% of the cases variant translocations involved 22q12 but with a chromosome(s) other than #11. The breakpoint on chromosome 22q12 appears to be the most consistently observed event in 92% of the cases, whereas, the breakpoint at chromosome 11q24 was observed in 88% of the cases.     

Spontaneous expression of fra(11)(q23) in a patient with Ewing's sarcoma and t(11;22)(q23;q11)

Cytogenetic analysis of a Ewing's sarcoma revealed a 46,XX,t(8;18)(q11;q21.3), t(11;22)(q23–24;q11–12) chromosome pattern. Observation of t(11;22) is consistent with other reported cases of Ewing's sarcoma. One breakpoint in this translocation, 11q23, coincides with the location of a folate-sensitive fragile site. Examination of peripheral blood leukocyte chromosomes from the patient revealed a 46,XX chromosome pattern with spontaneous, fluorodeoxyuridine-, and Bactrim-induced expression of fra(11)(q23). This may be the first demonstration of constitutional fra(11)(q23) expression in a patient with a neoplasm that exhibits a chromosome rearrangement involving this breakpoint and the first observation of spontaneous expression of this fragile site. These results provide a basis for discussion of the relationship between fragile sites and chromosome rearrangements.     

The role of cytogenetics in the classification of soft tissue tumours

 Soft tissue tumours represent a heterogeneous group of mesenchymal lesions, and their classification is the subject of continuous debate. Chromosome analysis, molecular cytogenetics and molecular assays may become increasingly useful in diagnosis, and this review summarises advances in the cytogenetic characterisation and classification of soft tissue tumours. Among the group of fibrous lesions, superficial fibromatosis exhibits trisomy 8. This genomic change is also observed in desmoid fibromatosis in association with trisomy 20. Trisomy 11 is the most frequently observed chromosomal aberration in congenital fibrosarcoma. Dermatofibrosarcoma protuberans and giant cell fibroblastoma share a translocation t(17;22), which supports the concept of the existence of a common differentiation pathway. Adipose tissue tumours is the group in which integration of genetics and pathology has been most fruitful. Ordinary lipomas cytogenetically show an abnormal karyotype in about half the cases. Genomic changes of the 11q13 region are observed in hibernoma. Lipoblastoma exhibits a specific 8q rearrangement in 8q11-q13. Loss of material from the region 16q13-qter and 13q deletions are observed in spindle cell/pleomorphic lipomas. The well-differentiated liposarcoma/atypical lipoma group is characterised karyotypically by the presence of one extra ring and/or extra giant chromosome marker. Myxoid and round cell liposarcoma share the same characteristic chromosome change: t(12;16)(q13;p11) in most cases. In the group of smooth muscle lesions most data are derived from uterine leiomyomas, which can be subclassified cytogenetically into seven different types. Half of all leiomyomas are chromosomally normal; the other half have one of six possible consistent chromosome changes. Alveolar rhabdomyosarcoma is characterised cytogenetically by two variant translocations t(2;13)(q35;q14) and t(1;13)(p36;q14). Among tenosynovial tumours, the localised type of giant cell tumour of tendon sheath exhibits two different karyotypic changes. One involves 1p11 in a translocation with chromosome 2 or with another chromosome. A second type involves 16q24. Synovial sarcoma is characterised cytogenetically by a translocation occurring between chromosome 18 and presumably two adjacent loci on the X chromosome. In neural tumours, abnormalities of chromosome 22 have been reported in benign schwannomas and perineuriomas. Malignant peripheral nerve sheath tumours exist in two main forms: sporadic and associated with the NF-1 syndrome. Karyotypes are very complex, but chromosomes 17q and 22q are very often involved. Clear cell sarcoma is characterised cytogenetically and molecularly by a translocation t(12;22)(q13;q12). The Ewing’s sarcoma/peripheral neuroectodermal tumour category shows a central karyotypic anomaly represented by the translocation t(11;22). The two variants t(21;22) and t(7;22) are found in some cases. Among cartilaginous lesion, the most frequently described anomaly is the t(9;22)(q22;q12) in extraskeletal myxoid chondrosarcoma. Intra-abdominal desmoplastic small round cell tumour is characterised by a t(11;22)(p13;q12). Received: 5 February 1997 / Accepted: 24 February 1997     

Trisomy 8 in primary esthesioneuroblastoma.

Esthesioneuroblastoma is a rare malignancy believed to be derived from neuroectodermal stem cells within the olfactory epithelium. We have obtained the karyotype of a primary esthesioneuroblastoma following brief (7-day) in vitro culture, and have determined that the only observable cytogenetic anomaly is the presence of an additional chromosome 8. Previously, the karyotypes of two cell lines established from metastatic esthesioneuroblastomas have been reported to contain the equivalent of three copies of chromosome 8, in addition to other chromosomal aberrations, including the reciprocal translocation, t(11;22)(q24;q12). Examination of the cytogenetic literature suggests that an extra copy of chromosome 8 is a common occurrence in undifferentiated small round cell tumors frequently observed to carry the t(11;22), including esthesioneuroblastoma, Ewing's sarcoma, peripheral neuroepithelioma, Askin's tumor, and rhabdomyosarcoma. These data, combined with our report of a small round cell tumor with the karyotype 47,XY, +8, indicate that trisomy 8 may be a common phenomenon in these tumors, and may also provide some sort of selective advantage to these tumor types.     

Detection of a novel reciprocal t(16;22)(q11.2;q12) in a Ewing sarcoma

Several structural and numerical chromosomal abnormalities have been identified as primary and secondary chromosomal aberrations in Ewing sarcoma (ES). The majority of these are t(11;22) and trisomies, especially of chromosome 8. Specific chromosomal abnormalities often correlate with particular morphologic or phenotypic subtypes of tumor and play an important role in prognosis. The objective of this report is the cytogenetic evaluation of a case of ES using G-banding, fluorescence in situ hybridization, and spectral karyotyping techniques. Multiple chromosomal aberrations were identified including a novel reciprocal t(16;22)(q11.2;q12).     

Frequency and Implications of Chromosome 8 and 12 Gains in Ewing Sarcoma

Ewing sarcoma (ES) is the second most common primary malignant tumor of bone in children and young adolescents. Most ES contain a pathognomonic translocation t(11;22)(q24;q12) that is likely a pivotal event in the tumorigenesis of these neoplasms. Many ES also contain nonrandom, numerical chromosomal aberrations, the most common of which are trisomies 8 and 12. In this study we evaluated the hypothesis that these trisomies might occur during neoplastic progression and might be associated with differences in biologic behavior. We tested this hypothesis using a combined cytogenetic and dual color fluorescence in situ hybridization approach to determine chromosome 8 and 12 copy number in 52 ES. Relative gains, primarily trisomies, of chromosomes 8 and 12 were found in 24 (46%) and 17 (33%) cases, respectively. Trisomy 8 and trisomy 12 were independent events acquired in a flexible order during ES genetic progression. Our preliminary findings also suggest a higher frequency of trisomies 8 and 12 in relapses than in primary tumors. Prospective studies will be required to determine whether either trisomy is prognostic in newly-diagnosed ES.     

Primitive neuroectodermal tumor of the myocardium: A case report, review of the literature, immunohistochemical, and ultrastructural study

We report the case of a primitive neuroectodermal tumor (PNET) arising in the heart of a 63-year-old man. The neuroectodermal nature of this tumor was confirmed by the immunohistochemical positivity for 013 (CD99) (the p30/32^MIC2 gene product) neuron specific enolase (monoclonal and polyclonal), synaptophysin and vimentin. Other markers, such as actin, desmin, myoglobin, chromogranin, keratin, and leukocyte common antigen were negative. The diagnosis was made on an endomyocardial biopsy and was confirmed in sections from the myocardial tumor found within the heart excised during cardiac transplant. Primitive neuroectodermal tumors have been reported in a variety of sites, most commonly in the extremities. No case has ever been reported within the myocardium, although one has been reported in the pericardium. In addition to morphological similarities, PNET and extraskeletal Ewing's sarcoma have been shown to possess the same chromosomal translocation, t11; 22, and the same cell surface antigen, P 30/32. Separation of this case from extraskeletal Ewing's sarcoma was possible because of the absence of PAS positivity, as well as the immunohistochemical positivity for at least two neural markers, as extraskeletal Ewing's sarcoma is only positive for neuron specific enolase.     

In vitro proliferative advantage of bone marrow cells with tetrasomy 8 in Ewing sarcoma

We describe a case of a 14.5-year-old boy with a clinically aggressive pelvic Ewing sarcoma. The tumor cells showed the presence of a typical t(11;22)(q24;q12) aberration and gains of chromosomes 8, 10, 14, and 21. To determine the size of the trisomy and tetrasomy 8 clones an interphase analysis by fluorescence in situ hybridization with a centromere-specific chromosome 8 probe was performed. Significant quantitative differences between metaphase and interphase data were obtained. It was shown that culturing of bone marrow cells leads to enrichment of tetrasomy 8 population that may be explained by the proliferative advantage of the tetrasomy 8 cells.     

Expression of a new human THY-1 related antigen in Ewing's sarcoma and peripheral neuroectodermal tumors

Ewing's sarcoma (ES), peripheral neuroectodermal tumor (PNET) and Askin's tumor of the chest wall share a reciprocal chromosomal translocation between the long arms of chromosomes 11 and 22 (q23-24; q12). In the absence of other distinguishing features this specific translocation is regarded as marker of a common and neuroectodermal origin for these rare tumors. A monoclonal antibody (HBA-71) developed in our laboratory has been found to recognize an unique ES and PNET associated antigen, which is also expressed in some normal tissues, including thymus, bone marrow, islets of Langerhans, ependyma and adenohypophysis. It is shown in this study that this HBA-71 antigen is closely related to the murine THY-1 antigens, major cell surface glycoproteins of thymocytes and brain in mice and rat. Both antigens have similar molecular ratios (18,000), amino acid compositions and sensitivity to tryptic digestion, show high cell surface expression, and binding of the appropriate antibodies to HBA-71 antigen triggers proliferation in thymocytes. The HBA-71 epitope may represent a primitive neuroectodermal marker of ES/PNET, or its expression may be directly linked to the reciprocal translocation invariably associated with HBA-71-positive ES and PNET tumors, which maps to the same region of chromosome 11 (q23-24) as the human Thy-1 gene.