An ultrasensitive method for detection of cell-free RNA

Monica C. Nesselbush, Bogdan A. Luca, Young-Jun Jeon, Isabel Jabara, Catherine B. Meador, Andrea Garofalo, Michael S. Binkley, Angela B. Hui, Iris van ‘t Erve, Nova Xu, William Y. Shi, Kevin J. Liu, Takeshi Sugio, Noah Kastelowitz, Emily G. Hamilton, Chih Long Liu, Mari Olsen, Rene F. Bonilla, Yi Peng Wang, Alice Jiang, Brianna Lau, Jordan Eichholz, Mandeep Banwait, Joseph Schroers-Martin, Jan Boegeholz, Daniel A. King, Helen Luikart, Mohammad S. Esfahani, Mahya Mehrmohamadi, Henning Stehr, Tyler Raclin, Robert Tibshirani, Kiran Khush, Sandy Srinivas, Helena Yu, Angela J. Rogers, Viswam S. Nair, James M. Isbell, Bob T. Li, Zofia Piotrowska, Lecia V. Sequist, Aaron N. Hata, Joel W. Neal, Heather A. Wakelee, Andrew J. Gentles, Ash A. Alizadeh () and Maximilian Diehn ()
Additional contact information
Monica C. Nesselbush: Stanford University
Bogdan A. Luca: Stanford University
Young-Jun Jeon: Sungkyunkwan University
Isabel Jabara: Stanford University
Catherine B. Meador: Massachusetts General Hospital Cancer Center
Andrea Garofalo: Stanford University
Michael S. Binkley: Stanford University
Angela B. Hui: Stanford University
Iris van ‘t Erve: Stanford University
Nova Xu: Stanford University
William Y. Shi: Stanford University
Kevin J. Liu: Stanford University
Takeshi Sugio: Stanford University
Noah Kastelowitz: Stanford University
Emily G. Hamilton: Stanford University
Chih Long Liu: Stanford University
Mari Olsen: Stanford University
Rene F. Bonilla: Stanford University
Yi Peng Wang: Stanford University
Alice Jiang: Stanford University
Brianna Lau: Stanford University
Jordan Eichholz: Memorial Sloan Kettering Cancer Center
Mandeep Banwait: Massachusetts General Hospital Cancer Center
Joseph Schroers-Martin: Stanford University
Jan Boegeholz: Stanford University
Daniel A. King: Stanford University
Helen Luikart: Stanford University
Mohammad S. Esfahani: Stanford University
Mahya Mehrmohamadi: Stanford University
Henning Stehr: Stanford University
Tyler Raclin: Stanford University
Robert Tibshirani: Stanford University
Kiran Khush: Stanford University
Sandy Srinivas: Stanford University
Helena Yu: Memorial Sloan Kettering Cancer Center
Angela J. Rogers: Stanford University
Viswam S. Nair: Fred Hutchinson Cancer Center
James M. Isbell: Memorial Sloan Kettering Cancer Center
Bob T. Li: Memorial Sloan Kettering Cancer Center
Zofia Piotrowska: Massachusetts General Hospital Cancer Center
Lecia V. Sequist: Massachusetts General Hospital Cancer Center
Aaron N. Hata: Massachusetts General Hospital and Harvard Medical School
Joel W. Neal: Stanford University
Heather A. Wakelee: Stanford University
Andrew J. Gentles: Stanford University
Ash A. Alizadeh: Stanford University
Maximilian Diehn: Stanford University

Nature, 2025, vol. 641, issue 8063, 759-768

Abstract: Abstract Sensitive methods for detection of cell-free RNA (cfRNA) could facilitate non-invasive gene expression profiling and monitoring of diseases1–6. Here we describe RARE-seq (random priming and affinity capture of cfRNA fragments for enrichment analysis by sequencing), a method optimized for cfRNA analysis. We demonstrate that platelet contamination can substantially confound cfRNA analyses and develop an approach to overcome it. In analytical validations, we find RARE-seq to be approximately 50-fold more sensitive for detecting tumour-derived cfRNA than whole-transcriptome RNA sequencing (RNA-seq), with a limit of detection of 0.05%. To explore clinical utility, we profiled 437 plasma samples from 369 individuals with cancer or non-malignant conditions and controls. Detection of non-small-cell lung cancer expression signatures in cfRNA increased with stage (6 out of 20 (30%) in stage I; 5 out of 8 (63%) in stage II; 10 out of 15 (67%) in stage III; 80 out of 96 (83% sensitivity) in stage IV at 95% specificity) and RARE-seq was more sensitive than tumour-naive circulating tumour DNA (ctDNA) analysis. In patients with EGFR-mutant non-small-cell lung cancer who developed resistance to tyrosine kinase inhibitors, we detected both histological transformation and mutation-based resistance mechanisms. Finally, we demonstrate the potential utility of RARE-seq for determination of tissue of origin, assessing benign pulmonary conditions and tracking response to mRNA vaccines. These results highlight the potential value of ultrasensitive cfRNA analysis and provide proof of concept for diverse clinical applications.

Date: 2025
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DOI: 10.1038/s41586-025-08834-1

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