Creytens David, van Gorp Joost, Ferdinande Liesbeth, Speel Ernst-Jan, Libbrecht Louis
*Department of Pathology, Ghent University and Ghent University Hospital, Ghent, Belgium †Department of Pathology, Diakonessenhuis Utrecht, Utrecht ‡Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.
Appl Immunohistochem Mol Morphol. 2015 Feb;23(2):126-33. doi: 10.1097/PDM.0000000000000041.
In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on paraffin-embedded material to test for other diagnostically, prognostically, or therapeutically relevant genomic mutations in lipomatous tumors.
在本研究中，使用多重连接依赖探针扩增技术（MLPA）——一种基于PCR的技术，对脂肪性软组织肿瘤中MDM2和CDK4扩增进行检测，并与荧光原位杂交（FISH）进行比较。对77例福尔马林固定、石蜡包埋的脂肪性肿瘤（27例良性脂肪肿瘤、28例非典型脂肪瘤/高分化脂肪肉瘤、18例去分化脂肪肉瘤和4例多形性脂肪肉瘤）进行了这两种技术的评估。使用MLPA，以>2的截断比值作为标准，71个样本中的36个（22例非典型脂肪瘤/高分化脂肪肉瘤和14例去分化脂肪肉瘤）显示MDM2和CDK4扩增。以FISH作为金标准，MLPA检测脂肪性软组织肿瘤中MDM2和CDK4扩增的灵敏度为90%（36/40），特异性为100%（31/31）。在高水平扩增（MDM2 - CDK4/CEP12比值>5）的情况下，一致性为100%。FISH检测到4例非典型脂肪瘤/高分化脂肪肉瘤（4/26，15%）MDM2和CDK4扩增水平较低（MDM2 - CDK4/CEP12比值在2至2.5之间），MLPA检测显示无扩增，不过MLPA检测到MDM2和CDK4有增益（比值在1.6至1.9之间）。在良性脂肪肿瘤和多形性脂肪肉瘤中未检测到扩增。此外，FISH和MLPA获得的比值之间一致性非常高。总之，MLPA被证明是一种用于筛查脂肪性肿瘤中MDM2/CDK4扩增的合适且直接的技术，尤其是在选择了正确的截断值和参考样本时，并且可以被认为是FISH的良好替代方法，用于确定脂肪肉瘤中的MDM2和CDK4扩增。此外，由于MLPA作为一种多重技术，能够同时检测多个感兴趣的染色体变化，未来它可能成为一种非常可靠且快速的分子分析方法，用于检测石蜡包埋材料中脂肪性肿瘤其他诊断、预后或治疗相关的基因组突变。
