Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples

Iolanda Capone; Fabio Bozzi; Gian Paolo Dagrada; Paolo Verderio; Elena Conca; Adele Busico; Maria Adele Testi; Valentina Monti; Matteo Duca; Claudia Proto; Silvia Damian; Alberta Piccolo; Federica Perrone; Elena Tamborini; Andrea Devecchi; Paola Collini; Daniele Lorenzini; Andrea Vingiani; Luca Agnelli; Giancarlo Pruneri

doi:10.37349/etat.2022.00102

Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples

Explor Target Antitumor Ther. 2022;3(5):582-597. doi: 10.37349/etat.2022.00102. Epub 2022 Oct 27.

Authors

Iolanda Capone^#¹, Fabio Bozzi^#¹, Gian Paolo Dagrada¹, Paolo Verderio², Elena Conca¹, Adele Busico¹, Maria Adele Testi¹, Valentina Monti¹, Matteo Duca³, Claudia Proto³, Silvia Damian³, Alberta Piccolo¹, Federica Perrone¹, Elena Tamborini¹, Andrea Devecchi¹, Paola Collini¹, Daniele Lorenzini¹, Andrea Vingiani^{1

4}, Luca Agnelli^{1

3}, Giancarlo Pruneri^{1

4}

Affiliations

¹ Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy.
² Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy.
³ Department of Medical Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133 Milan, Italy.
⁴ Department of Oncology and Hemato-oncology, University of Milan, 20133 Milan, Italy.

^# Contributed equally.

Abstract

Aim: Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics.

Methods: Herein, NGS data from 190 soft tissue tumors (STTs) and carcinoma cases, discussed in the context of the institutional Molecular Tumor Board, are reported and analyzed by FusionPlex^© Solid tumor kit through the manufacturer's pipeline and by two well-known fast and accurate open-source tools [Arriba (ARR) and spliced transcripts alignment to reference (STAR)-fusion (SFU)].

Results: The combination of FusionPlex^© Solid tumor with ArcherDX^® Analysis suite (ADx) analysis package has been proven to be sensitive and specific in STT samples, while partial loss of sensitivity has been found in carcinoma specimens.

Conclusions: Albeit ARR and SFU showed lower sensitivity, the use of additional fusion-detection tools can contribute to reinforcing or extending the output obtained by ADx, particularly in the case of low-quality input data. Overall, our results sustain the clinical use of NGS for the detection of fusion transcripts in FFPE material.

Keywords: Next-generation sequencing (NGS); RNA-sequencing; fluorescence in situ hybridization (FISH); formalin-fixed paraffin-embedded (FFPE); gene fusions.