An ultrasensitive method for detection of cell-free RNA

Monica C Nesselbush^#^{1

2

3}, Bogdan A Luca^#⁴, Young-Jun Jeon^#⁵, Isabel Jabara^{1

2

6}, Catherine B Meador^{7

8}, Andrea Garofalo^{1

3

9}, Michael S Binkley^{1

2}, Angela B Hui^{1

2}, Iris van 't Erve^{1

2}, Nova Xu^{1

2}, William Y Shi^{1

2

3}, Kevin J Liu^{1

2

3}, Takeshi Sugio^{1

9}, Noah Kastelowitz^{1

2}, Emily G Hamilton^{1

2

3}, Chih Long Liu^{1

9}, Mari Olsen^{1

9}, Rene F Bonilla^{1

2}, Yi Peng Wang^{1

2}, Alice Jiang^{1

2}, Brianna Lau^{1

2}, Jordan Eichholz¹⁰, Mandeep Banwait^{7

8}, Joseph Schroers-Martin^{1

9

11}, Jan Boegeholz^{1

9}, Daniel A King⁹, Helen Luikart¹², Mohammad S Esfahani^{1

9}, Mahya Mehrmohamadi^{1

9}, Henning Stehr⁴, Tyler Raclin^{1

2}, Robert Tibshirani¹³, Kiran Khush¹², Sandy Srinivas⁹, Helena Yu¹⁴, Angela J Rogers¹⁵, Viswam S Nair^{16

17}, James M Isbell¹⁸, Bob T Li¹⁴, Zofia Piotrowska^{7

8}, Lecia V Sequist^{7

8}, Aaron N Hata^{8

19}, Joel W Neal^{1

9}, Heather A Wakelee^{1

9}, Andrew J Gentles⁴, Ash A Alizadeh^{20

21

22

23}, Maximilian Diehn^{24

25

26}

Affiliations

¹ Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
² Department of Radiation Oncology, Stanford University, Stanford, CA, USA.
³ Program in Cancer Biology, Stanford University, Stanford, CA, USA.
⁴ Department of Pathology, Stanford University, Stanford, CA, USA.
⁵ Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea.
⁶ Department of Biology, Stanford University, Stanford, CA, USA.
⁷ Massachusetts General Hospital Cancer Center, Boston, MA, USA.
⁸ Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
⁹ Division of Oncology, Department of Medicine, Stanford University, Stanford, CA, USA.
¹⁰ Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹¹ Division of Hematology, Department of Medicine, Stanford University, Stanford, CA, USA.
¹² Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, CA, USA.
¹³ Department of Biomedical Data Science, Stanford University, Stanford, CA, USA.
¹⁴ Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹⁵ Division of Pulmonary, Allergy & Critical Care Medicine, Department of Medicine, Stanford University, Stanford, CA, USA.
¹⁶ Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.
¹⁷ Division of Pulmonary, Critical Care & Sleep Medicine, University of Washington, Seattle, WA, USA.
¹⁸ Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹⁹ Krantz Family Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA.
²⁰ Stanford Cancer Institute, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²¹ Division of Oncology, Department of Medicine, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²² Division of Hematology, Department of Medicine, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²³ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²⁴ Stanford Cancer Institute, Stanford University, Stanford, CA, USA. diehn@stanford.edu.
²⁵ Department of Radiation Oncology, Stanford University, Stanford, CA, USA. diehn@stanford.edu.
²⁶ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. diehn@stanford.edu.

^# Contributed equally.

PMID: 40240612
DOI: 10.1038/s41586-025-08834-1

An ultrasensitive method for detection of cell-free RNA

Monica C Nesselbush et al. Nature. 2025 May.

. 2025 May;641(8063):759-768.

doi: 10.1038/s41586-025-08834-1. Epub 2025 Apr 16.

Authors

Affiliations

¹ Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
² Department of Radiation Oncology, Stanford University, Stanford, CA, USA.
³ Program in Cancer Biology, Stanford University, Stanford, CA, USA.
⁴ Department of Pathology, Stanford University, Stanford, CA, USA.
⁵ Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Republic of Korea.
⁶ Department of Biology, Stanford University, Stanford, CA, USA.
⁷ Massachusetts General Hospital Cancer Center, Boston, MA, USA.
⁸ Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
⁹ Division of Oncology, Department of Medicine, Stanford University, Stanford, CA, USA.
¹⁰ Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹¹ Division of Hematology, Department of Medicine, Stanford University, Stanford, CA, USA.
¹² Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, CA, USA.
¹³ Department of Biomedical Data Science, Stanford University, Stanford, CA, USA.
¹⁴ Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹⁵ Division of Pulmonary, Allergy & Critical Care Medicine, Department of Medicine, Stanford University, Stanford, CA, USA.
¹⁶ Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.
¹⁷ Division of Pulmonary, Critical Care & Sleep Medicine, University of Washington, Seattle, WA, USA.
¹⁸ Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹⁹ Krantz Family Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA.
²⁰ Stanford Cancer Institute, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²¹ Division of Oncology, Department of Medicine, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²² Division of Hematology, Department of Medicine, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²³ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. arasha@stanford.edu.
²⁴ Stanford Cancer Institute, Stanford University, Stanford, CA, USA. diehn@stanford.edu.
²⁵ Department of Radiation Oncology, Stanford University, Stanford, CA, USA. diehn@stanford.edu.
²⁶ Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. diehn@stanford.edu.

^# Contributed equally.

PMID: 40240612
DOI: 10.1038/s41586-025-08834-1

Abstract

Sensitive methods for detection of cell-free RNA (cfRNA) could facilitate non-invasive gene expression profiling and monitoring of diseases^1-6. 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.

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Conflict of interest statement

Competing interests: E.G.H. has served as a compensated consultant for Foresight Diagnostics. K.K. has served as a scientific advisor to CareDx. H.Y. has received research support from AstraZeneca, Cullinan, Daiichi, Janssen, Blueprint, Lilly, Novartis and Pfizer. V.S.N. has received grant funding from Roche and Biodesix; and has served as a compensated consultant for Freenome, Verathon and Eurofins Viracor. J.M.I. has served on the advisory board of AstraZeneca and Merck; and has received research support from Foresight Diagnostics, Guardant Health and Invitae. B.T.L. has served as an uncompensated advisor and consultant to Amgen, Genentech, Boehringer Ingelheim, Lilly, AstraZeneca and Daiichi Sankyo; has received research grants to his institution from Amgen, Genentech, AstraZeneca, Daiichi Sankyo, Lilly, Illumina, GRAIL, Guardant Health, Hengrui Therapeutics, MORE Health and Bolt Biotherapeutics; has received academic travel support from MORE Health and Jiangsu Hengrui Medicine; is an inventor on two institutional patents at MSK (US62/685,057 and US62/514,661); and has intellectual property rights as a book author at Karger Publishers and Shanghai Jiao Tong University Press. Z.P. has served as a compensated consultant or received honoraria from Black Diamond Therapeutics, Janssen, Boehringer Ingelheim, Taiho, AstraZeneca, Blueprint Medicines, Daiichi Sankyo, Merck, Bayer, Sanofi, Takeda, Eli Lilly, Cullinan Oncology, C4 Therapeutics, Jazz Pharmaceuticals, Genentech and Guardant Health; has received research support (to institution) from Novartis, Takeda, Spectrum, AstraZeneca, Tesaro/GSK, Cullinan Oncology, Daiichi Sankyo, AbbVie, Janssen and Blueprint Medicines; has received travel support from AstraZeneca and Janssen; has received honoraria for educational programmes (CME) from Plexus, PeerView, Research to Practice, Clinical Care Options, Ology, CEC, PER, Haymarket Medical Education, Georgetown and Philips Gilmore Oncology; has received other honoraria from DAVA Oncology, Aptitude Health, University of Arkansas, Curio Science, Taiwan Society of Thoracic Surgeons, Medscape/WebMD, OncLive and Targeted Oncology. L.V.S. has received institutional funding for clinical trials from AstraZeneca, Novartis and Delfi Diagnostics. A.N.H. has received grants and/or research support from Amgen, Blueprint Medicines, BridgeBio, Bristol Myers Squibb, C4 Therapeutics, Eli Lilly, Novartis, Nuvalent, Pfizer, Roche/Genentech and Scorpion Therapeutics; and has served as a compensated consultant for Amgen, Engine Biosciences, Nuvalent, Oncovalent, Pfizer, TigaTx and Tolremo Therapeutics. J.W.N. has served in a consulting and/or advisory role for AstraZeneca, Genentech/Roche, Exelixis, Takeda Pharmaceuticals, Eli Lilly and Company, Amgen, Iovance Biotherapeutics, Blueprint Pharmaceuticals, Regeneron Pharmaceuticals, Natera, Sanofi/Regeneron, D2G Oncology, Surface Oncology, Turning Point Therapeutics, Mirati Therapeutics, Gilead Sciences, AbbVie, Summit Therapeutics, Novartis, Novocure, Janssen Oncology and Anheart Therapeutics; has received research support from Genentech/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Nektar Therapeutics, Takeda Pharmaceuticals, Adaptimmune, GSK, Janssen, AbbVie and Novocure; has received royalties from Up To Date; and has received honoraria for educational programmes (CME) from CME Matters, Clinical Care Options, Research to Practice, Medscape, Biomedical Learning Institute, MLI Peerview, Prime Oncology, Projects in Knowledge, Rockpointe, MJH Life Sciences, Medical Educator Consortium and HMP Education. H.A.W. has received institutional clinical trial support from AstraZeneca, Bayer, Bristol Myers Squibb, Genentech/Roche, Helsinn, Merck, SeaGen and Xcovery; has served as an uncompensated consultant to Genentech/Roche, Merck and AstraZeneca; and has contracted with Mirati. A.A.A. has received research support from Celgene and Bristol Myers Squibb; has served as a compensated consultant to Roche, Genentech, Celgene, Bristol Myers Squibb, FortySeven, Gilead, ADCT, Jannsen, CARGO, Foresight Diagnostics, Adaptive Biotech and Chugai; has served on the scientific advisory board for CiberMed and Foresight Diagnostics; and reports ownership interest in CiberMed, FortySeven, CARGO and Foresight Diagnostics. M.D. has received research support from AstraZeneca, Illumina and Varian Medical Systems; has received honoraria from Novartis and Bristol Myers Squibb Japan; has served as a compensated consultant to AstraZeneca, Boehringer Ingelheim, Genentech, Gritstone Bio, Illumina, Regeneron and Roche; and reports ownership interest in CiberMed, Foresight Diagnostics and Gritstone Bio. M.C.N., B.A.L., M.D. and A.A.A. also report patent filings related to cancer and non-malignant biomarkers in cfRNA (PCT/US2024/029513). The remaining authors declare no competing interests.

References

1. Larson, M. H. et al. A comprehensive characterization of the cell-free transcriptome reveals tissue- and subtype-specific biomarkers for cancer detection. Nat. Commun. 12, 2357 (2021). - PubMed - PMC - DOI
1. Chen, S. et al. Cancer type classification using plasma cell-free RNAs derived from human and microbes. eLife 11, e75181 (2022). - PubMed - PMC - DOI
1. Morlion, A. et al. Patient-specific alterations in blood plasma cfRNA profiles enable accurate classification of cancer patients and controls. Preprint at medRxiv https://doi.org/10.1101/2023.05.24.23290388 (2023).
1. Roskams-Hieter, B. et al. Plasma cell-free RNA profiling distinguishes cancers from pre-malignant conditions in solid and hematologic malignancies. NPJ Precis. Oncol. 6, 28 (2022). - PubMed - PMC - DOI
1. Zhu, Y. et al. Integrative analysis of long extracellular RNAs reveals a detection panel of noncoding RNAs for liver cancer. Theranostics 11, 181–193 (2021). - PubMed - PMC - DOI

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An ultrasensitive method for detection of cell-free RNA

Affiliations

An ultrasensitive method for detection of cell-free RNA

Authors

Affiliations

Abstract

Conflict of interest statement

References

MeSH terms

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LinkOut - more resources

Full Text Sources

Research Materials

Miscellaneous