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. 2022 Sep 20;23(19):11007.
doi: 10.3390/ijms231911007.

mRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase

Anneleen Decock  1   2 , David Creytens  2   3   4 , Steve Lefever  1   2 , Joni Van der Meulen  1   2   5 , Jasper Anckaert  1   2 , Ariane De Ganck  6 , Jill Deleu  1   2 , Bram De Wilde  1   2   7 , Carolina Fierro  6 , Scott Kuersten  8 , Manuel Luypaert  6 , Isabelle Rottiers  2   3 , Gary P Schroth  8 , Sandra Steyaert  6 , Katrien Vanderheyden  1 , Eveline Vanden Eynde  1   2 , Kimberly Verniers  1   2 , Joke Verreth  1 , Jo Van Dorpe  2   3   4 , Jo Vandesompele  1   2   6
Affiliations

mRNA Capture Sequencing and RT-qPCR for the Detection of Pathognomonic, Novel, and Secondary Fusion Transcripts in FFPE Tissue: A Sarcoma Showcase

Anneleen Decock et al. Int J Mol Sci. .

Abstract

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.

Keywords: RT-qPCR; alveolar rhabdomyosarcoma; formalin-fixed paraffin-embedded (FFPE) tissue; fusion gene; fusion transcript; mRNA capture sequencing; sarcoma; undifferentiated round cell sarcoma.

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Conflict of interest statement

C.F. is an employee, A.D.G., M.L. and S.S. are former employees, and J.V. (Jo Vandesompele) is co-founder and CSO at Biogazelle, a CRO providing RNA sequencing services. G.S. and S.K. are employees of Illumina, providing the TruSight RNA Pan-Cancer Panel. The other authors declare no potential conflict of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study.

Figures

Figure 1
Figure 1
mRNA capture sequencing and RT-qPCR for the detection of pathognomonic, novel, and secondary fusion transcripts in FFPE sarcoma tissue. Cohort I, comprising six patients with a known pathognomonic fusion, is profiled using the TruSight RNA Pan-Cancer Panel. These data validated the mRNA capture sequencing analysis workflow for the identification of fusion transcripts. Subsequently, a second cohort of sarcomas that were designated fusion gene-negative by FISH analysis was analyzed using TruSeq RNA Exome sequencing. Multiple pathognomonic, novel, and secondary fusion transcripts were picked up and confirmed by RT-qPCR.
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