Dataset Information

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

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.

SUBMITTER: Decock A

PROVIDER: S-EPMC9569610 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Publications

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

Decock Anneleen A Creytens David D Lefever Steve S Van der Meulen Joni J Anckaert Jasper J De Ganck Ariane A Deleu Jill J De Wilde Bram B Fierro Carolina C Kuersten Scott S Luypaert Manuel M Rottiers Isabelle I Schroth Gary P GP Steyaert Sandra S Vanderheyden Katrien K Vanden Eynde Eveline E Verniers Kimberly K Verreth Joke J Van Dorpe Jo J Vandesompele Jo J

International journal of molecular sciences 20220920 19

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 Exo ...[more]

PMID: 36232302

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Project description:Rapid diagnosis of acute promyelocytic leukemia (APL) with promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa) contributes to a highly effective therapy with all-trans retinoic acid (ATRA). Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is a valuable tool to diagnose APL with PML-RARa. However, a single RT-qPCR analysis, which is laborious and costly, has to be performed in three reactions to determine whether one of the three PML-RARa transcripts is present and to quantify the involved transcript. This paper describes a novel TaqMan MGB probe-based 3-plex RT-qPCR assay in a single reaction to detect simultaneously the three PML-RARa transcripts. Specific primers and probe were designed, and the results were further normalized to the Abelson gene. The detection results for the serially diluted plasmid indicate that the analytical sensitivity was 10 copies per reaction for PML-RARa bcr1, bcr2, and bcr3. A relatively high sensitivity of 10-4 was achieved with this assay when analyzing the bcr1 transcripts obtained from the NB4 cell line. The reproducibility was satisfactory because the coefficients of variation of cycle threshold values were less than 3% for both inter- and intra-assays. After testing 319 newly diagnosed patients with leukemia (including 61 APL cases), the results of the 3-plex RT-qPCR assay completely agreed with the traditional methods used for the detection of PML-RARa. The quantitative results of the 3-plex RT-qPCR were highly correlated with the single RT-qPCR and showed similar assay sensitivity for 60 PML-RARa positive APL samples at diagnosis and 199 samples from 57 patients during follow-up. Interestingly, one PML-RARa bcr2 case at diagnosis with breakpoint at 1579, which was not detected by the single RT-q-PCR, was detected by the 3-plex RT-qPCR assay. The 3-plex RT-qPCR assay is a specific, sensitive, stable, and cost-effective method that can be used for the rapid diagnosis and treatment monitoring of APL with PML-RARa.

Dataset Information

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

Publications

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

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