5.A transcription assay for EWS oncoproteins in Xenopus oocytes.
King Pan NG ; Felix CHEUNG ; Kevin A W LEE
Protein & Cell 2010;1(10):927-934
Aberrant chromosomal fusion of the Ewing's sarcoma oncogene (EWS) to several different cellular partners produces the Ewing's family of oncoproteins (EWS-fusion-proteins, EFPs) and associated tumors (EFTs). EFPs are potent transcriptional activators, dependent on the N-terminal region of EWS (the EWS-activation-domain, EAD) and this function is thought to be central to EFT oncogenesis and maintenance. Thus EFPs are promising therapeutic targets, but detailed molecular studies will be pivotal for exploring this potential. Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function. Therefore, the lack of manipulatable assays for the EAD presents a significant barrier to progress. Using Xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient, bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.
Animals
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Biological Assay
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Female
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Oncogene Proteins
;
genetics
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Oncogenes
;
genetics
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Oocytes
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metabolism
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pathology
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RNA-Binding Protein EWS
;
genetics
;
metabolism
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Sarcoma, Ewing
;
genetics
;
pathology
;
Xenopus
6.EWSR1-SMAD3 positive fibroblastic tumor: a clinicopathological analysis.
Hai Yan SU ; Lu ZHAO ; Gang JI ; Qian Lan YAO ; Qian Ming BAI ; Xiao Yan ZHOU ; Jian WANG
Chinese Journal of Pathology 2023;52(1):19-24
Objective: To investigate the clinicopathological features, immunophenotypes and molecular genetics of EWSR1-SMAD3 positive fibroblastic tumor (ESFT) with an emphasis on differential diagnosis. Methods: The clinicopathological data, immunohistochemical profiles and molecular profiles of 3 ESFT cases diagnosed at the Department of Pathology, Fudan University Shanghai Cancer Center from 2018 to 2021were analyzed. The related literature was also reviewed. Results: There were two males and one female. The patients were 24, 12 and 36 years old, respectively. All three tumors occurred in the subcutis of the foot with the disease duration of 6 months to 2 years. The tumors were presented with a slowly growing mass or nodule, accompanied with pain in 1 patient. The tumors ranged in size from 0.1 to 1.6 cm (mean, 1.0 cm). Microscopically, the tumors were located in the subcutaneous tissue with a nodular or plexiform growth pattern. They were composed of cellular fascicles of bland spindle cells with elongated nuclei and fine chromatin. One of the tumors infiltrated into adjacent adipose tissue. There was no nuclear atypia or mitotic activities. All three tumors showed prominent stromal hyalinization with zonal pattern present in one case. Focal punctate calcification was noted in two cases. The immunohistochemical studies showed that tumor cells were diffusely positive for ERG and negative for CD31 and CD34, with Ki-67 index less than 2%. Fluorescence in situ hybridization on the two tested cases identified EWSR1 gene rearrangement. The next generation sequencing analysis demonstrated EWSR1-SMAD3 fusion in all three cases. During the follow up, one patient developed local recurrence 24 months after the surgery. Conclusions: ESFT is a benign fibroblastic neoplasm and has a predilection for the foot, characterized by ERG immunoreactivity and EWSR1-SMAD3 fusion. Local recurrence might occur when incompletely excised. Familiarity with its clinicopathological features is helpful in distinguishing it from other spindle cell neoplasms that tend to occur at acral sites.
Adult
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Child
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Female
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Humans
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Male
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Biomarkers, Tumor/analysis*
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China
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In Situ Hybridization, Fluorescence
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Neoplasms, Fibrous Tissue/pathology*
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RNA-Binding Protein EWS/genetics*
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Smad3 Protein/genetics*
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Soft Tissue Neoplasms/surgery*
7.Detection of chromosomal translocation in fresh samples of myxoid/round cell liposarcoma by long-distance polymerase chain reaction.
Hua XIANG ; Jian WANG ; Masanori HISAOKA ; Xiong-zeng ZHU
Chinese Journal of Pathology 2007;36(6):412-413
Adult
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Base Sequence
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DNA, Neoplasm
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genetics
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Exons
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Female
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Humans
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Liposarcoma
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genetics
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Liposarcoma, Myxoid
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genetics
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Male
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Middle Aged
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Molecular Sequence Data
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Oncogene Proteins, Fusion
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genetics
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Polymerase Chain Reaction
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methods
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RNA-Binding Protein EWS
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genetics
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RNA-Binding Protein FUS
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genetics
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Sequence Analysis, DNA
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Transcription Factor CHOP
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genetics
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Translocation, Genetic
8.Solid variant of angiomatoid fibrous histocytoma:report of 3 cases.
Zheng WANG ; Qin-he FAN ; Jian WANG ; Yong-ling DING
Chinese Journal of Pathology 2013;42(11):744-747
OBJECTIVETo study the clinicopathologic features, immunophenotype, molecular genetics and differential diagnosis of solid variant of angiomatoid fibrous histocytoma.
METHODSThe clinicopathologic features of 3 cases of solid variant of angiomatoid fibrous histocytoma were analyzed and the literature was reviewed.
RESULTSThere were a total of 2 males and 1 female. The age of patients ranged from 9 to 12 years. The patients presented with a painless mass located in left forearm, left knee or back. The lesions were treated by complete surgical resection. On gross examination, the tumors varied from 1.6 cm to 4.5 cm in greatest dimension. They were well-circumscribed and had pale yellow to grayish-red solid cut surface. Histologically, the tumor was composed of histocytoid cells arranged in sheet-like pattern. A fibrous pseudocapsule surrounded by lymphocytes and plasma cells was identified. Immunohistochemical study showed that the tumor cells in all cases were positive for vimentin and CD68. They were negative for S100 protein, cytokeratin, CD34, CD31, smooth muscle actin, CD35, CD21 and CD30. Two cases also expressed CD99 and one of them was positive for desmin and epithelial membrane antigen. Fluorescence in-situ hybridization was positive for EWSR1 gene.
CONCLUSIONSSolid type represents a variant of angiomatoid fibrous histocytoma and is considered as tumor of borderline malignant potential. Definitive diagnosis requires thorough histologic examination and clinical correlation. Immunohistochemistry and EWSR1 gene study are helpful in further delineation and differential diagnosis. Complete resection or wide local excision with post-operative follow up is the main modality of treatment.
Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Back ; Calmodulin-Binding Proteins ; genetics ; Child ; Dendritic Cell Sarcoma, Follicular ; metabolism ; pathology ; Diagnosis, Differential ; Female ; Forearm ; Histiocytoma, Malignant Fibrous ; genetics ; metabolism ; pathology ; surgery ; Humans ; Knee ; Male ; Neoplasms, Muscle Tissue ; pathology ; Neurilemmoma ; metabolism ; pathology ; RNA-Binding Protein EWS ; RNA-Binding Proteins ; genetics ; Soft Tissue Neoplasms ; genetics ; metabolism ; pathology ; surgery ; Vimentin ; metabolism
9.Multiplex RT-PCR assay for detecting fusion genes of soft tissue small round cell tumors using paraffin-embedded and formalin-fixed tissue.
Yan QI ; Bin CHANG ; Li-juan PANG ; Chun-xia LIU ; Wen-hao HU ; Hong-an LI ; Jin-fang JIANG ; Jian-feng GAO ; Jing-yu WEI ; Feng LI
Chinese Journal of Pathology 2006;35(10):634-636
Base Sequence
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Formaldehyde
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chemistry
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Humans
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Molecular Sequence Data
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Oncogene Proteins, Fusion
;
genetics
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Paraffin Embedding
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Proto-Oncogene Protein c-fli-1
;
genetics
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RNA, Neoplasm
;
genetics
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metabolism
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RNA-Binding Protein EWS
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Reverse Transcriptase Polymerase Chain Reaction
;
methods
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Rhabdomyosarcoma
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genetics
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Sarcoma, Ewing
;
genetics
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Sarcoma, Synovial
;
genetics
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Soft Tissue Neoplasms
;
genetics
;
Tissue Fixation
10.Molecular detection of EWS-Ets fusion transcripts and their clinicopathologic significance in Ewing's sarcoma/peripheral primitive neuroectodermal tumor.
Hua WANG ; Jie ZHENG ; Yu-ping WANG ; Yu YANG ; Jiang-feng YOU
Chinese Medical Journal 2005;118(16):1323-1329
BACKGROUNDEwing's sarcoma/peripheral primitive neuroectodermal tumor (ES/pPNET) is often difficult to distinguish from other small round cell tumors. The EWS-Ets gene fusions that result from chromosomal translocations in this tumor provide potential molecular diagnostic markers. To apply these molecular markers to commonly available archival materials, we evaluated the feasibility of detecting EWS-Ets including EWS-Fli1 and EWS-ERG fusion transcripts in paraffin-embedded tissues and its diagnostic value for detecting ES/pPNET.
METHODSThirteen paraffin-embedded samples of ES/pPNETs were retrieved from archives. Thirteen cases of other tumors with small round cell features (including rhabdomyosarcoma, neuroblastoma, lymphoma, small cell carcinoma, and desmoplastic small round cell tumor) were used as negative controls. Beta-actin and beta2-microglobulin were used as internal controls. A nested reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay was performed to detect the EWS-Fli1 and EWS-ERG fusion transcripts.
RESULTSBeta-actin and beta2-microglobulin were detected in 10/13 and 13/13 ES/pPNETs, respectively. EWS-Fli1 fusion transcripts were detected in 11 of 13 (85%) ES/pPNETs. Three chimeric transcripts, all EWS-Fli1, were detected in ES/pPNET samples. Among 11 EWS-Fli1-positive cases, 7 cases had a type I fusion transcript involving fusion of EWS exon 7 with Fli1 exon 6, 2 cases had a type II fusion transcript involving EWS exon 7 with Fli1 exon 5, and 2 cases expressed fusion transcripts involving EWS exon 7 and Fli1 exon 8. Type I EWS-Fli1 fusion predominated over other types. Fusion types could not be distinguished in the remaining 2 cases. Thirteen negative controls did not show detectable chimeric messages. There was a significant relationship between EWS-Fli1 fusion transcripts and CD99 expression.
CONCLUSIONSMolecular detection of EWS-Fli1 fusion transcripts in formalin-fixed paraffin-embedded material by nested RT-PCR is feasible and is useful for the diagnosis and differential diagnosis of ES/pPNETs.
Adolescent ; Adult ; Child ; Female ; Humans ; Male ; Middle Aged ; Neuroectodermal Tumors, Primitive, Peripheral ; genetics ; pathology ; Oncogene Proteins, Fusion ; genetics ; Polymerase Chain Reaction ; Proto-Oncogene Protein c-fli-1 ; RNA, Messenger ; analysis ; RNA-Binding Protein EWS ; Sarcoma, Ewing ; genetics ; pathology ; Transcription Factors ; genetics