Unannotated small RNA clusters associated with circulating extracellular vesicles detect early stage liver cancer
Johann von Felden 1 2 , Teresa Garcia-Lezana 1 , Navneet Dogra 3 4 , Edgar Gonzalez-Kozlova 3 , Mehmet Eren Ahsen 3 , Amanda Craig 1 , Stacey Gifford 4 , Benjamin Wunsch 4 , Joshua T Smith 4 , Sungcheol Kim 4 , Jennifer E L Diaz 3 , Xintong Chen 3 , Ismail Labgaa 1 5 , Philipp Haber 1 , Reena Olsen 6 , Dan Han 6 , Paula Restrepo 3 7 , Delia D'Avola 1 8 , Gabriela Hernandez-Meza 1 , Kimaada Allette 3 , Robert Sebra 3 9 , Behnam Saberi 1 , Parissa Tabrizian 10 11 , Amon Asgharpour 1 , Douglas Dieterich 1 , Josep M Llovet 1 12 13 , Carlos Cordon-Cardo 3 6 , Ash Tewari 14 , Myron Schwartz 11 , Gustavo Stolovitzky 15 4 , Bojan Losic 15 16 17 , Augusto Villanueva 18 19
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.