Meier V S, Kühne T, Jundt G, Gudat F
Institute for Pathology, University of Basel, Switzerland.
Diagn Mol Pathol. 1998 Feb;7(1):29-35. doi: 10.1097/00019606-199802000-00006.
Most Ewing tumors (ET), including Ewing sarcomas, peripheral primitive neuroectodermal tumors (PNET), and Askin's tumors, can be defined according to the specific chromosomal translocations t(11;22)(q24;q12) (EWS-FLI-1) or t(21;22)(q21;q12) (EWS-ERG). Detection of the chimeric RNA transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) has greatly facilitated the diagnosis of ET. Because of variable chromosomal breakpoint locations, however, the EWS gene fusions with FLI-1 and ERG genes are highly heterogenous, resulting in different sizes of the amplification products. To improve the diagnostic usefulness of the RT-PCR assay, we have developed an assay to detect chimeric mRNA transcripts by nested RT-PCR, followed by digestion of the PCR fragments with three different restriction endonucleases. This allows confirmation of the specificity of the PCR product and provides a rapid method to determine the combination of exons present in a transcript. In the 12 Ewing tumors tested, five different exon combinations were detected. In nine repeat biopsies of four patients, the case-specific translocation remained unchanged. One additional central PNET had no ET-specific translocation. In conclusion, the suggested combination of RT-PCR and restriction analysis of the PCR products allows a rapid and specific determination of ET-specific translocations.
大多数尤因肿瘤（ET），包括尤因肉瘤、外周原始神经外胚层肿瘤（PNET）和阿斯金肿瘤，可根据特定的染色体易位t(11;22)(q24;q12)（EWS-FLI-1）或t(21;22)(q21;q12)（EWS-ERG）来定义。通过逆转录聚合酶链反应（RT-PCR）检测嵌合RNA转录本极大地促进了ET的诊断。然而，由于染色体断点位置的变化，EWS基因与FLI-1和ERG基因的融合高度异质，导致扩增产物大小不同。为提高RT-PCR检测的诊断效用，我们开发了一种通过巢式RT-PCR检测嵌合mRNA转录本的方法，随后用三种不同的限制性内切酶消化PCR片段。这可以确认PCR产物的特异性，并提供一种快速方法来确定转录本中存在的外显子组合。在所检测的12例尤因肿瘤中，检测到五种不同的外显子组合。在4例患者的9次重复活检中，病例特异性易位保持不变。另外1例中枢性PNET没有ET特异性易位。总之，建议的RT-PCR与PCR产物限制性分析相结合的方法能够快速、特异性地确定ET特异性易位。
