我们评估了mRNA捕获测序的性能，以鉴定不同肉瘤类型的FFPE组织中的融合转录本，然后进行RT-qPCR确认。要验证我们的工作流程，使用TruSightRNA泛癌症小组分析了6种具有特定染色体重排的阳性对照肿瘤.FusionCatcher的融合转录物调用证实了这些畸变，并能够鉴定融合基因伴侣和断点。接下来,全转录组TruSeqRNA外显子组测序应用于17个融合基因阴性的肺泡横纹肌肉瘤（ARMS）或未分化的圆形细胞肉瘤（URCS）肿瘤，荧光原位杂交(FISH)未能鉴定出经典的病理学重排.对于六个病人来说，很容易检测到病态融合转录本，即，两名ARMS患者的PAX3-FOXO1，和EWSR1-FLI1，EWSR1-ERG，或EWSR1-NFATC2在四名URCS患者中。对于剩下的11名患者，通过RT-qPCR确认了11个新鉴定的融合转录本，包括COPS3-TOM1L2，NCOA1-DTNB，WWTR1-LINC01986,PLAA-MOB3B,ARMS患者的AP1B1-CHEK2和BRD4-LEUTX融合转录本。此外,在诊断为EWSR1-NFATC2阳性肉瘤的患者中反复检测到的次级融合转录本得到证实(COPS4-TBC1D9,PICALM-SYTL2,SMG6-VPS53和UBE2F-ALS2).总之,这项研究表明，mRNA捕获测序提高了病理基因融合的检出率，并能够鉴定肉瘤中的新的和次级融合转录本。
We assess the performance of mRNA capture sequencing to identify fusion transcripts in FFPE tissue of different sarcoma types, followed by RT-qPCR confirmation. To validate our workflow, six positive control tumors with a specific chromosomal rearrangement were analyzed using the TruSight RNA Pan-Cancer Panel. Fusion transcript calling by FusionCatcher confirmed these aberrations and enabled the identification of both fusion gene partners and breakpoints. Next, whole-transcriptome TruSeq RNA Exome sequencing was applied to 17 fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS) tumors, for whom fluorescence in situ hybridization (FISH) did not identify the classical pathognomonic rearrangements. For six patients, a pathognomonic fusion transcript was readily detected, i.e., PAX3-FOXO1 in two ARMS patients, and EWSR1-FLI1, EWSR1-ERG, or EWSR1-NFATC2 in four URCS patients. For the 11 remaining patients, 11 newly identified fusion transcripts were confirmed by RT-qPCR, including COPS3-TOM1L2, NCOA1-DTNB, WWTR1-LINC01986, PLAA-MOB3B, AP1B1-CHEK2, and BRD4-LEUTX fusion transcripts in ARMS patients. Additionally, recurrently detected secondary fusion transcripts in patients diagnosed with EWSR1-NFATC2-positive sarcoma were confirmed (COPS4-TBC1D9, PICALM-SYTL2, SMG6-VPS53, and UBE2F-ALS2). In conclusion, this study shows that mRNA capture sequencing enhances the detection rate of pathognomonic fusions and enables the identification of novel and secondary fusion transcripts in sarcomas.
