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

Abstract

ObjectiveSurveillance 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.DesignEVs 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.ResultsExRNA 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.ConclusionThese 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.

Hepatocellular carcinoma surveillance: current practice and future directions

“…Similar to ctDNA, EV and its cargoes are regarded as promising biomarkers to augment current HCC surveillance methods due to their early presence in circulation [81] . Our recent phase 2 biomarker study applied an HCC EV Digital Scoring Assay to quantify the identified 10 HCC-specific mRNA markers [74] from purified HCC EVs and showed the resultant digital scores could noninvasively detect BCLC stage 0-A HCC from cirrhosis with an AUROC of 0.93 (sensitivity = 94%, specificity = 89%) [82] . Currently, almost all of the existing EV-based biomarker studies are still at phase II (case-control) [81,83] , further phase III (prospective specimen collection, retrospective blinded evaluation), and phase IV studies (prospective cohort) are required to validate their performance for detecting early-stage HCC [80] .…”

Biomarker Development Using Liquid Biopsy in Hepatocellular Carcinoma

“…60 EVs are promising biomarkers for early HCC detection evidenced by the increasing number of studies published. [61][62][63][64][65] As key mediators of intercellular communication EVs are present in the circulation at early stages of disease. 66,67 One study in 158 patients with HCC that a 10-gene molecular signature had 94% sensitivity and 89% specificity in detecting early HCC.…”

“…62 von Felden et al identified a 3-small RNA clusters signature overexpressed in plasma exosomal RNA of 157 patients with HCC (p < 0.01). 63 This biomarker was then independently validated in phase 2 case-control studies in 209 patients (including 105 with early stage HCC) and demonstrated 86% sensitivity and 91% specificity in detection of early HCC from controls at risk with AUC 0.87, independent of AFP (composite model including AFP increased AUC to 0.93). 63 Regarding the role of CTCs in early detection, a recent meta-analysis of 20 studies showed utility in CTC for diagnosis of HCC.…”

“…63 This biomarker was then independently validated in phase 2 case-control studies in 209 patients (including 105 with early stage HCC) and demonstrated 86% sensitivity and 91% specificity in detection of early HCC from controls at risk with AUC 0.87, independent of AFP (composite model including AFP increased AUC to 0.93). 63 Regarding the role of CTCs in early detection, a recent meta-analysis of 20 studies showed utility in CTC for diagnosis of HCC. 68 As the numbers of CTCs in early-stage tumors are low, these may play a more limited role in early detection.…”

Open Problems in Extracellular RNA Data Analysis: Insights From an ERCC Online Workshop

“…Bojan Losic presented a method for the discovery of small RNA clusters (smRCs, pronounced smirks) expressed in circulating liver cancer EVs ( von Felden et al, 2021 ). The method is de novo , not relying on mapping reads to annotated RNAs.…”