Objective: To investigate the clinical application of EWSR1 gene rearrangement by fluorescence in situ hybridization (FISH) in bone and soft tissue tumors and to analyze the cases with atypical signal pattern. Methods: The cases detected for EWSR1 gene rearrangement by FISH in Beijing Jishuitan Hospital, Capital Medical University from 2014 to 2021 were collected, and the value of detecting EWSR1 gene rearrangement for diagnosing bone and soft tissue tumors was analyzed. The cases with atypical positive signals were further analyzed by next generation sequencing (NGS). Results: FISH using EWSR1 break-apart probe kit was successfully performed in 97% (205/211) of cases, 6 cases failed. Four of the 6 failures were due to improper decalcification, 1 case due to signal overlap caused by thick slices, and 1 case due to signal amplification and disorder. EWSR1 gene rearrangements were positive in 122 cases (122/205, 59%), atypical positive signal in 8 cases (8/205, 4%), and negative in 75 cases (75/205, 37%). In cases testing positive, the percentage of positive cells ranged from 34% to 98%, with 120 cases (120/122, 98%) showing a positive cell percentage greater than 50%. Among the 205 successfully tested cases, 156 cases were histologically diagnosed as Ewing's sarcoma, of which 110 were positive (110/156, 71%), 7 were atypical positive (7/156, 4%), and 39 were negative (39/156, 25%). Nine cases were histologically diagnosed as clear cell sarcoma of soft tissue, of which 6 were positive (6/9), 1 was atypical positive (1/9), and 2 were negative (2/9). Five cases were histologically diagnosed as extraskeletal myxoid chondrosarcoma, of which 2 were positive (2/5) and 3 were negative (3/5). Three cases were histologically diagnosed as angiomatoid fibrous histiocytoma, of which 2 were positive (2/3) and 1 was negative (1/3). Two cases were histologically diagnosed as myoepithelioma of soft tissue, of which 1 was positive (1/2) and 1 was negative (1/2). One case was histologically diagnosed as olfactory neuroblastoma with a positive result. The 29 other tumor cases including osteosarcoma, synovial sarcoma, and malignant melanoma and others were all negative. Basing on histology as the standard for diagnosis and considering atypical positive cases as negative, comparing with the 29 cases of other tumors as control group, the sensitivity for diagnosing Ewing's sarcoma through the detection of EWSR1 gene rearrangement was 71%, and the specificity was 100%; the sensitivity for diagnosing clear cell sarcoma of soft tissue was 67%, and the specificity was 100%; the sensitivity for diagnosing extraskeletal myxoid chondrosarcoma was 40%, and the specificity was 100%; the sensitivity for diagnosing angiomatoid fibrous histiocytoma was 67%, and the specificity was 100%; the sensitivity for diagnosing myoepithelioma of soft tissue was 50%, and the specificity was 100%; the sensitivity for diagnosing olfactory neuroblastoma was 100%, and the specificity was 100%. Four of 8 cases with atypical positive signals analyzed by NGS showed EWSR1 rearrangement, including EWSR1::FLI1 in one case of Ewing sarcoma, EWSR1::NFATC2 in one case of EWSR1::NFATC2-rearranged sarcoma, EWSR1::ATF1 in one case of clear cell sarcoma of soft tissue and EWSR1::NR4A3 in one case of extraskeletal myxoid chondrosarcoma. Conclusions: Detection of EWSR1 rearrangement by FISH is of utmost significance in the diagnosis of bone and soft tissue tumors. Cases with atypical positive signals should be further scrutinized, correlating with their histomorphology and verifying by NGS if necessary.
目的： 探讨应用荧光原位杂交（FISH）检测骨与软组织肿瘤EWSR1基因重排的临床应用价值并对出现不典型信号的病例进行分析。 方法： 收集首都医科大学附属北京积水潭医院2014—2021年应用FISH检测EWSR1基因重排的病例，分析其用于诊断相关骨与软组织肿瘤的临床应用价值并对FISH检测结果呈不典型信号的病例进行二代测序以进一步分析。 结果： 应用FISH检测EWSR1基因重排病例211例，检测成功205例（205/211，97%），失败6例（6/211，3%）。6例检测失败病例中，4例因骨组织脱钙不当未检测到荧光信号，1例因切片太厚信号重叠无法判读，1例因信号扩增且杂乱无法判读。205例检测成功病例中，阳性122例（122/205，59%），不典型阳性8例（8/205，4%），阴性75例（75/205，37%）。阳性者其阳性细胞百分数为34%~98%，其中120例（120/122，98%）阳性细胞百分数>50%。205例检测成功病例中，156例组织学诊断为尤因肉瘤，其中阳性110例（110/156，71%），不典型阳性7例（7/156，4%），阴性39例（39/156，25%）。9例组织学诊断为软组织透明细胞肉瘤，其中阳性6例（6/9），不典型阳性1例（1/9），阴性2例（2/9）。5例组织学诊断为骨外黏液样软骨肉瘤，其中阳性2例（2/5），阴性3例（3/5）。3例组织学诊断为血管瘤样纤维组织细胞瘤，其中阳性2例（2/3），阴性1例（1/3）。2例组织学诊断为软组织肌上皮瘤，其中阳性1例（1/2），阴性1例（1/2）。1例组织学诊断为嗅神经母细胞瘤，结果为阳性。检测其他肿瘤29例，包括骨肉瘤、滑膜肉瘤、恶性黑色素瘤等，均为阴性。以组织学为金标准，将不典型阳性按阴性考虑，以29例其他肿瘤为对照组病例，检测EWSR1基因重排诊断尤因肉瘤的灵敏度为71%，特异度为100%；诊断软组织透明细胞肉瘤的灵敏度为67%，特异度为100%；诊断骨外黏液样软骨肉瘤的灵敏度为40%，特异度为100%；诊断血管瘤样纤维组织细胞瘤的灵敏度为67%，特异度为100%；诊断软组织肌上皮瘤的灵敏度为50%，特异度为100%；诊断嗅神经母细胞瘤的灵敏度为100%，特异度为100%。8例具有不典型阳性信号的病例中4例有足够组织材料送检二代测序，结果均证实存在EWSR1基因重排，形成的融合基因分别为EWSR1：：FLI1（尤因肉瘤）、EWSR1：：NFATC2（EWSR1：：NFATC2肉瘤）、EWSR1：：ATF1（软组织透明细胞肉瘤）和EWSR1：：NR4A3（骨外黏液样软骨肉瘤）。 结论： 应用FISH检测EWSR1基因重排对骨与软组织肿瘤的诊断具有重要意义。对于出现不典型信号病例的解读要紧密结合组织形态，必要时应用其他分子检测方法验证。.