Nishio Jun, Althof Pamela A, Bailey Jacqueline M, Zhou Ming, Neff James R, Barr Frederic G, Parham David M, Teot Lisa, Qualman Stephen J, Bridge Julia A
Department of Pathology and Microbiology, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, 68198-3135, USA.
Lab Invest. 2006 Jun;86(6):547-56. doi: 10.1038/labinvest.3700416.
A valuable diagnostic adjunct and important prognostic parameter in alveolar rhabdomyosarcoma (ARMS) is the identification of translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), and the associated PAX3-FKHR and PAX7-FKHR fusion transcripts, respectively. Most RMS fusion gene type studies have been based on reverse transcriptase-polymerase chain reaction (RT-PCR) detection of the fusion transcript, a technique limited by RNA quality and failure of devised primer sets to detect unusual variants. As an alternative approach, we developed a fluorescence in situ hybridization (FISH) assay that can: (1) distinguish between the two most common ARMS-associated fusion genes; (2) identify potential unusual variant translocations; (3) assess histologic components in mixed alveolar/embryonal RMS; and (4) be performed on paraffinized tissue. FISH analyses of 75 specimens (40 ARMS, 16 ERMS, 8 mixed ARMS/ERMS, and 11 non-RMS tumors) using selected cosmid clone, bacterial, P1-derived, and yeast artificial chromosome probe sets were successful in all but two cases. Among specimens with informative results for both FISH and RT-PCR or standard karyotyping, PAX/FKHR classification results were concordant in 94.6% (53/56). The three discordant cases included one exhibiting a t(2;13) by FISH that was subsequently confirmed by repeat RT-PCR, a second showing a rearrangement of the PAX3 locus only (consistent with the presence of a PAX3 variant translocation), and a third revealing a t(2;13) by FISH that lacked this translocation cytogenetically. Both alveolar and embryonal components of the mixed ARMS/ERMS subtype were negative for PAX3, PAX7, and FKHR rearrangements, a surprising finding confirmed by RT-PCR and/or conventional karyotyping. These data demonstrate that FISH with newly designed probe sets is a reliable and highly specific method of detecting t(1;13) and t(2;13) in routinely processed tissue and may be useful in differentiating ARMS from other small round cell tumors. The findings also suggest that FISH may be a more sensitive assay than RT-PCR in some settings, capable of revealing variant translocations.
在肺泡横纹肌肉瘤（ARMS）中，一个有价值的诊断辅助手段和重要的预后参数是分别鉴定t(2;13)(q35;q14)和t(1;13)(p36;q14)易位以及相关的PAX3-FKHR和PAX7-FKHR融合转录本。大多数横纹肌肉瘤融合基因类型研究基于逆转录聚合酶链反应（RT-PCR）检测融合转录本，该技术受RNA质量限制，且设计的引物组无法检测到异常变体。作为一种替代方法，我们开发了一种荧光原位杂交（FISH）检测方法，它能够：（1）区分两种最常见的与ARMS相关的融合基因；（2）识别潜在的异常变体易位；（3）评估混合性肺泡/胚胎性横纹肌肉瘤中的组织学成分；（4）在石蜡包埋组织上进行检测。使用选定的黏粒克隆、细菌人工染色体、P1衍生人工染色体和酵母人工染色体探针组对75个标本（40个ARMS、16个胚胎性横纹肌肉瘤、8个混合性ARMS/胚胎性横纹肌肉瘤和11个非横纹肌肉瘤肿瘤）进行FISH分析，除两例以外均成功。在FISH和RT-PCR或标准核型分析结果均有意义的标本中，PAX/FKHR分类结果的一致性为94.6%（53/56）。三例不一致的病例包括：一例FISH显示t(2;13)，随后经重复RT-PCR证实；第二例仅显示PAX3基因座重排（与存在PAX3变体易位一致）；第三例FISH显示t(2;13)，但细胞遗传学上缺乏这种易位。混合性ARMS/胚胎性横纹肌肉瘤亚型的肺泡和胚胎成分PAX3、PAX7和FKHR重排均为阴性，这一惊人发现经RT-PCR和/或传统核型分析证实。这些数据表明，使用新设计的探针组进行FISH是在常规处理组织中检测t(1;13)和t(2;13)的可靠且高度特异的方法，可能有助于将ARMS与其他小圆细胞肿瘤区分开来。研究结果还表明，在某些情况下，FISH可能比RT-PCR更敏感，能够揭示变体易位。
