Objective: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma.
Methods: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features.
Results: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis.
Conclusion: Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.
目的: 探讨荧光原位杂交(fluorescence in situ hybridization，FISH)法检测MDM2和DDIT3基因在各亚型脂肪肉瘤的临床诊断价值。
方法: 回顾性分析北京大学第一医院病理科2015年1月至2019年12月诊断的62例脂肪肉瘤的临床病理学特征，所有病例进行了MDM2基因扩增FISH检测，其中48例进行了DDIT3基因断裂FISH检测，观察MDM2和DDIT3基因信号改变模式，并综合临床病理学特征确诊不同亚型脂肪肉瘤。
结果: 62例患者以男性多见(男: 女=1.75:1)，中位年龄59(31~89)岁。脂肪肉瘤组织学亚型包括20例非典型脂肪瘤性肿瘤/高分化脂肪肉瘤(atypical lipomatous tumour/well differentiated liposarcoma，ALT/WDLPS)、26例去分化脂肪肉瘤(dedifferentiated liposarcoma, DDLPS)、13例黏液样脂肪肉瘤(myxoid liposarcoma, MLPS)和3例多形性脂肪肉瘤(pleomorphic liposarcoma, PLPS)，其中23例(23/26)DDLPS和8例(8/20)WDLPS位于腹膜后，而MLPS和PLPS则均不位于腹膜后。肿瘤发生部位在不同亚型脂肪肉瘤间的差异具有统计学意义(P < 0.05)。组织形态学上，各亚型脂肪肉瘤均可见多少不等的黏液样间质，且差别具有统计学意义(P < 0.05)。MDM2基因扩增见于所有ALT/WDLPS和DDLPS(均为100%，20/20及26/26)；DDIT3分离探针典型信号表现为基因断裂/易位，仅见于MLPS(100%，13/13)，但值得注意的是，该探针还可出现非典型信号(端粒侧信号簇状扩增)，比较常见，包括绝大多数DDLPS(96.2%, 25/26)和ALT/WDLPS(83.3%，5/6，探针只随机检测6例)。对照分析探针说明书上的设计图，DDIT3分离探针之红绿色探针并未覆盖DDIT3基因本身，而是跨越了DDIT3基因，且探针端粒侧红色探针覆盖了CDK4基因，而DDIT3基因与CDK4基因距离很近，因此，当DDIT3分离探针端粒侧出现簇状扩增信号，提示CDK4基因扩增而非DDIT3基因断裂。随机挑选10例出现这种非典型信号的病例，以FISH法进一步应用CDK4和DDIT3计数探针进行验证，证实为CDK4基因扩增(100%，10/10)，其中2例同时检测到DDIT3基因扩增(20%，2/10)；DDIT3分离探针还存在另一种少见的非典型信号，即基因拷贝数增加(黄色融合信号数目≥3个)，对应形态学表现为多形性和预后差，见于1例DDLPS和2例PLPS。
结论: 利用FISH技术观察MDM2和DDIT3基因信号改变模式时, 需要注意对非典型信号做出正确判读，并结合患者的临床病理学特征，才能对不同亚型脂肪肉瘤做出正确诊断，进一步指导临床治疗。
Keywords: DDIT3 gene; In situ hybridization, fluorescence; Liposarcoma; MDM2 gene.