Abstract 402: RNA-sequencing of plasma exosomes reveals specific transcriptomic profiles in patients with hepatocellular carcinoma
Authors: Felden Johann von , Craig Amanda J. , Ahsen Mehmet Eren , Labgaa Ismail , D'Avola Delia , Meza Gabriela Hernandez , Allette Kimaada , Dogra Navneet , Lezana Teresa Garcia , Tabrizian Parissa , Asgharpour Amon , Dieterich Douglas , Sebra Robert , Stolovitzky Gustavo , Schwartz Myron , Villanueva Augusto 
Published: August 2019

Abstract
Hepatocellular carcinoma (HCC) is the tumor with the highest increase in incidence and mortality in the last 20 years. Exosomes are nano-sized particles loaded with nucleic acids and proteins involved in cell-to-cell communication. The aims of this study are: 1) to establish a method to isolate exosomes from plasma of HCC patients, and 2) to detect de-regulated transcripts in exosomes from HCC patients compared to controls.
In this pilot study, we included 10 HCC patients and 5 controls at high-risk of HCC development (i.e. patients with cirrhosis and/or chronic hepatitis B). Plasma exosomes were isolated by differential ultracentrifugation. Presence of exosomes was confirmed by morphological assessment with electron microscopy, nanoparticle tracking analysis and immune-labeling of EV specific markers (CD9, CD63, CD81, and TSG101) with ExoView and Western Blotting. RNA from exosomes was extracted and quantified with Bioanalyzer. Library preparation was done with SMARTer smRNA-Seq kit and whole transcriptome sequencing was performed on an Illumina HiSeq2500 instrument. Differential gene expression was done with LIMMA and enrichment analyses (i.e., gene ontology) with Enrichr.
HCC patients and controls were matched for age (median 67 vs. 63 yrs.), sex (70% vs. 67% males), and etiology (viral 70% vs. 67%). Sequencing reads obtained from exosomes mapped mRNA, lincRNA and small RNA, including miRNAs and Y-RNAs. Compartment ontology enrichment analysis of the top 250 expressed genes matched to exosomes and membrane-bounded vesicles (FDR&lt;0.01). Genes from hepatic lineage were among the top expressed genes including ALB, BAAT, CRP, C3/5, FGA/B, CYP and APO family. This confirms the presence of liver-derived exosomes in human plasma. We identified 252 differentially expressed genes between HCC and controls (FDR&lt;0.05). Among them, we found genes with known aberrant expression in HCC tissue samples such as ARF1 (Fold-change [FC]: 5 fold up-regulated), CDKN2A (FC:-3.2), H3F3A (FC:8) or TAGLN2 (FC:7). Among the 35 highest differentially expressed genes (FC&gt;8) between HCC and controls, there was an enrichment in previously unreported Y-RNAs (37%, FDR&lt;0.001) and microRNAs (11%, FDR&lt;0.001).
We conclude that whole transcriptomic analysis of plasma exosomes is feasible in patients with HCC. Our pilot study detects significant differences in the transcriptomic profiles of HCC patients compared to controls, including an enrichment of upregulated miRNAs and Y-RNAs.
Citation Format: Johann von Felden, Amanda J. Craig, Mehmet Eren Ahsen, Ismail Labgaa, Delia D'Avola, Gabriela Hernandez Meza, Kimaada Allette, Navneet Dogra, Teresa Garcia Lezana, Parissa Tabrizian, Amon Asgharpour, Douglas Dieterich, Robert Sebra, Gustavo Stolovitzky, Myron Schwartz, Augusto Villanueva. RNA-sequencing of plasma exosomes reveals specific transcriptomic profiles in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 402.