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Original research
Unannotated small RNA clusters associated with circulating extracellular vesicles detect early stage liver cancer
  1. Johann von Felden1,2,
  2. Teresa Garcia-Lezana1,
  3. Navneet Dogra3,4,
  4. Edgar Gonzalez-Kozlova3,
  5. Mehmet Eren Ahsen3,
  6. Amanda Craig1,
  7. Stacey Gifford4,
  8. Benjamin Wunsch4,
  9. Joshua T Smith4,
  10. Sungcheol Kim4,
  11. Jennifer E L Diaz3,
  12. Xintong Chen3,
  13. Ismail Labgaa1,5,
  14. Philipp Haber1,
  15. Reena Olsen6,
  16. Dan Han6,
  17. Paula Restrepo3,7,
  18. Delia D'Avola1,8,
  19. Gabriela Hernandez-Meza1,
  20. Kimaada Allette3,
  21. Robert Sebra3,9,
  22. Behnam Saberi1,
  23. Parissa Tabrizian10,11,
  24. Amon Asgharpour1,
  25. Douglas Dieterich1,
  26. Josep M. Llovet1,12,13,
  27. Carlos Cordon-Cardo3,6,
  28. Ash Tewari14,
  29. Myron Schwartz11,
  30. Gustavo Stolovitzky3,4,
  31. Bojan Losic3,15,16,
  32. Augusto Villanueva1,17
  1. 1Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  2. 2Department of Medicine, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
  3. 3Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  4. 4IBM Thomas J Watson Research Center, Yorktown Heights, New York, USA
  5. 5Department of Visceral Surgery, Lausanne University Hospital, Lausanne, Switzerland
  6. 6Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  7. 7Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  8. 8Liver Unit and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Clínica Universidad de Navarra, Pamplona, Spain
  9. 9Sema4, Stamford, Connecticut, USA
  10. 10Recanati/Miller Transplantation Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  11. 11Department of Surgery, Mount Sinai School of Medicine, New York, New York, USA
  12. 12Liver Cancer Translational Research Laboratory, BCLC Group, IDIBAPS, CIBEREHD, Hospital Clinic, Universitat de Barcelona, Barcelona, Catalonia, Spain
  13. 13Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain
  14. 14Department of Urology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  15. 15Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  16. 16Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  17. 17Division of Hematology and Medical Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA
  1. Correspondence to Dr Augusto Villanueva, Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; augusto.villanueva{at}mssm.edu; Dr Gustavo Stolovitzky, Sema4, Stamford, CT 06902, USA; gustavo.stolovitzky{at}sema4.com; Dr Bojan Losic; blosic{at}gmail.com

Abstract

Objective Surveillance tools for early cancer detection are suboptimal, including hepatocellular carcinoma (HCC), and biomarkers are urgently needed. Extracellular vesicles (EVs) have gained increasing scientific interest due to their involvement in tumour initiation and metastasis; however, most extracellular RNA (exRNA) blood-based biomarker studies are limited to annotated genomic regions.

Design EVs were isolated with differential ultracentrifugation and integrated nanoscale deterministic lateral displacement arrays (nanoDLD) and quality assessed by electron microscopy, immunoblotting, nanoparticle tracking and deconvolution analysis. Genome-wide sequencing of the largely unexplored small exRNA landscape, including unannotated transcripts, identified and reproducibly quantified small RNA clusters (smRCs). Their key genomic features were delineated across biospecimens and EV isolation techniques in prostate cancer and HCC. Three independent exRNA cancer datasets with a total of 479 samples from 375 patients, including longitudinal samples, were used for this study.

Results ExRNA smRCs were dominated by uncharacterised, unannotated small RNA with a consensus sequence of 20 nt. An unannotated 3-smRC signature was significantly overexpressed in plasma exRNA of patients with HCC (p<0.01, n=157). An independent validation in a phase 2 biomarker case–control study revealed 86% sensitivity and 91% specificity for the detection of early HCC from controls at risk (n=209) (area under the receiver operating curve (AUC): 0.87). The 3-smRC signature was independent of alpha-fetoprotein (p<0.0001) and a composite model yielded an increased AUC of 0.93.

Conclusion These findings directly lead to the prospect of a minimally invasive, blood-only, operator-independent clinical tool for HCC surveillance, thus highlighting the potential of unannotated smRCs for biomarker research in cancer.

  • surveillance
  • cancer prevention
  • hepatobiliary cancer
  • gene expression
  • tumour markers

Data availability statement

Data are available in a public, open access repository. RNA-sequencing data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession numbers E-MTAB-8528 and E-MTAB-8529. Code availability statement: all code will be made publicly available at Dr Losic GitHub site.

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Data availability statement

Data are available in a public, open access repository. RNA-sequencing data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession numbers E-MTAB-8528 and E-MTAB-8529. Code availability statement: all code will be made publicly available at Dr Losic GitHub site.

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Footnotes

  • GS, BL and AV are joint senior authors.

  • Contributors Study design and drafting of the manuscript: JvF, GS, BL and AV. Sample collection: JvF, TG-L, ND, AC, IL, PH, DD'A, BS, PT, AA, DD, CC-C, AT, MS and AV. Experimental procedures: JvF, TG-L, ND, AC, SG, BW, JTS, SK, JELD, RO, DH, KA, RS, GS, BL and AV. Data analysis: JvF, TG-L, ND, EG-K, MEA, JELD, XC, PR, GH-M, JL, GS, BL and AV. All authors have critically revised the manuscript and gave their final approval.

  • Funding JvF is supported by the German Research Foundation (grant numbers: FE1746/1-1 and FE1746/3-1) and the Clinician Scientist Programme at University Medical Centre Hamburg. TG-L is supported by the Grant for Studies Broadening from the Spanish Association for the Study of the Liver (Asociación Española para el Estudio del Hígado, AEEH). AC is supported by the National Cancer Institute, Ruth L Kirschstein NRSA Institutional Research Training (grant number: CA078207). MEA, JELD, XC and BL were supported by the Icahn Institute of Genomics and Multiscale Biology. IL is supported by a grant from the Swiss National Science Foundation, from Foundation Roberto and Gianna Gonella and Foundation SICPA. PH is supported by the German Research Foundation (gran number: HA8754/1-1). DD'A is supported by the Grant for Studies Broadening from the Spanish Association for the Study of the Liver (AEEH) and the Cancer Research Grant from Nuovo Soldati Foundation. JML is supported by grants from the US Department of Defence (grant number: CA150272P3), European Commission Framework Programme 7 (HEPTROMIC, proposal number: 259744) and Horizon 2020 Programme (HEPCAR, proposal number: 6 67 273–2), the Asociación Española Contra el Cáncer (AECC), Samuel Waxman Cancer Research Foundation, Spanish National Health Institute (SAF2013-41027) and Grup de Recerca Consolidat–Recerca Translacional en Oncologia Hepàtica, AGAUR (Generalitat de Catalunya), SGR 1162. CC-C is supported by NCI (grant number: P01-CA087497) and NIH (grant number: U54-OD020353). AV is supported by the US Department of Defence (grant number: CA150272P3).

  • Competing interests JvF, BL and AV are inventors in a provisional patent application for the 3-smRC signature. JvF received advisory board fees from Roche. DD'A received consulting fees from Almylam and Novartis. JML is receiving research support from Bayer HealthCare Pharmaceuticals, Eisai Inc, Bristol-Myers Squibb, Boehringer-Ingelheim and Ipsen, and consulting fees from Eli Lilly, Bayer HealthCare Pharmaceuticals, Bristol-Myers Squibb, Eisai Inc, Celsion Corporation, Exelixis, Merck, Ipsen, Genentech, Roche, Glycotest, Nucleix, Sirtex, Mina Alpha Ltd and AstraZeneca. AV has received consulting fees from Boehringer Ingelheim, Guidepoint and Fujifilm; advisory board fees from Bristol-Myers Squibb, Genentech, Gilead, Nucleix and NGM Pharmaceuticals; and research support from Eisai Pharmaceuticals. The remaining authors have nothing to declare in relation to this manuscript.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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