mRNA capture sequencing and RT-qPCR for the detection of pathognomonic, novel, and secondary fusion transcripts in FFPE tissue: a sarcoma showcase

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.

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.

Publication
International Journal of Molecular Sciences
Anneleen Decock
Anneleen Decock
PostDoctoral Fellow

Working with extracellular RNA of liquid biopsies in cancer

Steve Lefever
Steve Lefever
PostDoctoral Fellow (01/2008-09/2020)
Jasper Anckaert
Jasper Anckaert
Bioinformatician

The real Jasper

Jill Deleu
Jill Deleu
PostDoctoral Fellow
Bram De Wilde
Bram De Wilde
PostDoctoral Fellow
Katrien Vanderheyden
Katrien Vanderheyden
Lab Technician
Eveline Vanden Eynde
Eveline Vanden Eynde
Lab Technician
Kimberly Verniers
Kimberly Verniers
Lab Technician
Jo Vandesompele
Jo Vandesompele
Professor

RNA addict trying to connect all the dots