Unannotated small RNA clusters associated with circulating extracellular vesicles detect early stage liver cancer

Johann Von Felden, Teresa Garcia-Lezana, Navneet Dogra, Edgar Gonzalez-Kozlova, Mehmet Eren Ahsen, Amanda Craig, Stacey Gifford, Benjamin Wunsch, Joshua T. Smith, Sungcheol Kim, Jennifer E.L. Diaz, Xintong Chen, Ismail Labgaa, Philipp Haber, Reena Olsen, Dan Han, Paula Restrepo, Delia D'Avola, Gabriela Hernandez-Meza, Kimaada AlletteRobert Sebra, Behnam Saberi, Parissa Tabrizian, Amon Asgharpour, Douglas Dieterich, Josep M. Llovet, Carlos Cordon-Cardo, Ash Tewari, Myron Schwartz, Gustavo Stolovitzky, Bojan Losic, Augusto Villanueva

Research output: Contribution to journalArticlepeer-review

24 Scopus citations


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.

Original languageEnglish
Pages (from-to)2069-2080
Number of pages12
Issue number10
StatePublished - 1 Oct 2022


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


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