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Volume 26
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10.3390/ijms262311300
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Accurate RET Fusion Detection in Solid Tumors Using RNA Sequencing Coverage Imbalance Analysis

by
Ivan Gaziev
1,†,
Anna Khristichenko
2,†,
Daniil Luppov
1,
Maria Suntsova
1,
Ekaterina Bondarenko
3,
Maria Reinberg
3,
Alina Matrosova
1,3,
Nadezhda Khilal
1,
Maksim Sorokin
1,4,
Marina Sekacheva
1,
Elena Poddubskaya
1,
Anton Buzdin
1,2,5 and
Galina Zakharova
1,*
1
Institute for Personalized Oncology, Biomedical Science & Technology Park, FSAEI HE I.M. Sechenov First Moscow State Medical University of MOH of Russia (Sechenovskiy University), 119991 Moscow, Russia
2
Department of Genetics and Life Sciences, Sirius University of Science and Technology, 354340 Sirius Federal Territory, Russia
3
National Medical Research Center for Endocrinology, 117292 Moscow, Russia
4
Oncobox LLC., 119991 Moscow, Russia
5
PathoBiology Group, European Organization for Research and Treatment of Cancer (EORTC), 1200 Brussels, Belgium
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(23), 11300; https://doi.org/10.3390/ijms262311300 (registering DOI)
Submission received: 16 October 2025 / Revised: 15 November 2025 / Accepted: 19 November 2025 / Published: 22 November 2025
(This article belongs to the Special Issue Molecular Diagnostics and Genomics of Tumors, 2nd Edition)
Versions Notes

Abstract

Accurate detection of oncogenic gene fusions is becoming increasingly important given the availability of highly effective targeted therapies. However, their identification in clinical practice remains challenging due to the rarity of individual events, diversity of partner genes, and variability of breakpoint locations. Conventional approaches such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) lack multiplexing capacity and demonstrate variable sensitivity and specificity, while direct identification of fusion transcripts in whole-transcriptome sequencing (RNA-seq) profiles provides broader applicability but limited sensitivity, as fusion junctions are frequently supported by a minimal number of reads or even no reads at all. In this study, a novel approach was employed to accurately detect clinically actionable RET (REarranged during Transfection) fusions. This approach entailed the measurement of the imbalance in RNA-seq read coverage of potential fusion oncogenes at their 3′ and 5′ exons. A total of 1327 experimental solid tumor RNA-seq profiles were screened, including 154 non-small cell lung cancer and 221 thyroid cancer samples. The RET status was validated in 78 selected cases by targeted NGS and Sanger sequencing. An analysis of the coverage imbalance was conducted, which enabled the accurate discrimination between true and false positive RET fusions. This approach outperformed other methods and yielded 100% sensitivity and specificity with optimized thresholds. The findings were validated using an independent cohort of 79 thyroid cancer cases, confirming the reliability of the results. Among the 18 RET fusion-positive samples, one was identified as an extremely rare case (RUFY3::RET), and two were determined to be novel fusions (FN1::RET, PPP1R21::RET). The findings of this study demonstrate that exon coverage imbalance analysis serves as a robust complement to computational RNA-seq analysis pipelines for the detection of clinically relevant RET fusions.
Keywords: fusion oncogene; receptor tyrosine kinase; Ret Proto-Oncogene (RET); RET rearrangement; RNA sequencing; tumor molecular diagnostics; clinical oncology fusion oncogene; receptor tyrosine kinase; Ret Proto-Oncogene (RET); RET rearrangement; RNA sequencing; tumor molecular diagnostics; clinical oncology

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MDPI and ACS Style

Gaziev, I.; Khristichenko, A.; Luppov, D.; Suntsova, M.; Bondarenko, E.; Reinberg, M.; Matrosova, A.; Khilal, N.; Sorokin, M.; Sekacheva, M.; et al. Accurate RET Fusion Detection in Solid Tumors Using RNA Sequencing Coverage Imbalance Analysis. Int. J. Mol. Sci. 2025, 26, 11300. https://doi.org/10.3390/ijms262311300

AMA Style

Gaziev I, Khristichenko A, Luppov D, Suntsova M, Bondarenko E, Reinberg M, Matrosova A, Khilal N, Sorokin M, Sekacheva M, et al. Accurate RET Fusion Detection in Solid Tumors Using RNA Sequencing Coverage Imbalance Analysis. International Journal of Molecular Sciences. 2025; 26(23):11300. https://doi.org/10.3390/ijms262311300

Chicago/Turabian Style

Gaziev, Ivan, Anna Khristichenko, Daniil Luppov, Maria Suntsova, Ekaterina Bondarenko, Maria Reinberg, Alina Matrosova, Nadezhda Khilal, Maksim Sorokin, Marina Sekacheva, and et al. 2025. "Accurate RET Fusion Detection in Solid Tumors Using RNA Sequencing Coverage Imbalance Analysis" International Journal of Molecular Sciences 26, no. 23: 11300. https://doi.org/10.3390/ijms262311300

APA Style

Gaziev, I., Khristichenko, A., Luppov, D., Suntsova, M., Bondarenko, E., Reinberg, M., Matrosova, A., Khilal, N., Sorokin, M., Sekacheva, M., Poddubskaya, E., Buzdin, A., & Zakharova, G. (2025). Accurate RET Fusion Detection in Solid Tumors Using RNA Sequencing Coverage Imbalance Analysis. International Journal of Molecular Sciences, 26(23), 11300. https://doi.org/10.3390/ijms262311300

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