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 Allette; Robert 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

doi:10.1136/gutjnl-2021-325036

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

Gut. 2021 Jul 28:gutjnl-2021-325036. doi: 10.1136/gutjnl-2021-325036. Online ahead of print.

Authors

Johann von Felden^{1

2}, Teresa Garcia-Lezana¹, Navneet Dogra^{3

4}, Edgar Gonzalez-Kozlova³, Mehmet Eren Ahsen³, Amanda Craig¹, Stacey Gifford⁴, Benjamin Wunsch⁴, Joshua T Smith⁴, Sungcheol Kim⁴, Jennifer E L Diaz³, Xintong Chen³, Ismail Labgaa^{1

5}, Philipp Haber¹, Reena Olsen⁶, Dan Han⁶, Paula Restrepo^{3

7}, Delia D'Avola^{1

8}, Gabriela Hernandez-Meza¹, Kimaada Allette³, Robert Sebra^{3

9}, Behnam Saberi¹, Parissa Tabrizian^{10

11}, Amon Asgharpour¹, Douglas Dieterich¹, Josep M Llovet^{1

12

13}, Carlos Cordon-Cardo^{3

6}, Ash Tewari¹⁴, Myron Schwartz¹¹, Gustavo Stolovitzky^{15

4}, Bojan Losic^{15

16

17}, Augusto Villanueva^{18

19}

Affiliations

¹ Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
² Department of Medicine, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.
³ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
⁴ IBM Thomas J Watson Research Center, Yorktown Heights, New York, USA.
⁵ Department of Visceral Surgery, Lausanne University Hospital, Lausanne, Switzerland.
⁶ Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
⁷ Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
⁸ Liver Unit and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Clínica Universidad de Navarra, Pamplona, Spain.
⁹ Sema4, Stamford, Connecticut, USA.
¹⁰ Recanati/Miller Transplantation Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
¹¹ Department of Surgery, Mount Sinai School of Medicine, New York, New York, USA.
¹² Liver Cancer Translational Research Laboratory, BCLC Group, IDIBAPS, CIBEREHD, Hospital Clinic, Universitat de Barcelona, Barcelona, Catalonia, Spain.
¹³ Institució Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain.
¹⁴ Department of Urology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
¹⁵ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York, USA augusto.villanueva@mssm.edu gustavo.stolovitzky@sema4.com blosic@gmail.com.
¹⁶ Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
¹⁷ Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
¹⁸ Division of Liver Diseases, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA augusto.villanueva@mssm.edu gustavo.stolovitzky@sema4.com blosic@gmail.com.
¹⁹ Division of Hematology and Medical Oncology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

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.

Keywords: cancer prevention; gene expression; hepatobiliary cancer; surveillance; tumour markers.

Abstract

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