EXTRACELLULAR VESICLE DNA-PK MRNA - A CANDIDATE LIQUID BIOMARKER FOR HEPATOCELLULAR CARCINOMA

Introduction

Hepatocellular carcinoma (HCC) deaths are rising, with late stage presentation and poor liver function limiting treatment options. Trans-arterial chemo-embolisation (TACE) can be used for intermediate stages but can cause harm and predictive biomarkers are needed. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is central to DNA damage repair and its overexpression in HCC biopsies is associated with resistance to TACE [Cornell et al. Clin Cancer Res 21, 925–933,2015]. Biopsy carries risks. Liquid biopsy tools as adjuncts to biopsy, for predictive and monitoring purposes, are needed. Previous attempts to robustly detect/quantify DNA-PK in serum have failed. Our aim is to explore mRNA transcripts in extracellular vesicles (EVs) as a liquid biopsy tool for HCC patients.

Methods

Here we have focused on mRNA expression in circulating cell-free RNA within EVs. Serum from healthy volunteers (HV)(n=6) and patients with chronic liver disease (CLD) with (n=10) and without HCC (n=6) was evaluated. EV mRNA was isolated using Qiagen ExoRNeasy Midi kits. Yields were improved by extended incubation and repeated elution. EV mRNA circulating transcripts levels of control (18S ribosomal, hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)), liver representative (albumin) and PRKDC (gene encoding DNA-PK) genes were analysed by qPCR.

Results

This method enabled the robust isolation of mRNA protected by EVs, including from frozen samples stored from patients with CLD and cancer. 18s was detected at much higher levels than HPRT or GAPDH in all cases, reflecting its abundance in EVs. Serum albumin was within the normal range for all cases, with albumin EV mRNA detected in all the samples at similar levels, confirming this liver-specific transcript can be efficiently isolated and detected. PRKDC EV mRNA was also detected in all cases, but at very low levels in 4/6 healthy volunteers. In contrast, 15/16 CLD +/- HCC had detectable PRKDC (p<0.05, Chi Square) with 2-10 fold elevations relative to HV, with some HCC patients having 20-25 fold elevations.

Conclusions

We have optimised a method for isolation of mRNA from serum EVs, confirming that liver specific, as well as candidate predictive HCC biomarkers, can be detected. Our ongoing work will define the relationships between candidate EV mRNA transcripts, stage of liver disease and stage of cancer, while also exploring a predictive role for response to DNA damaging therapies (like TACE). A DNA-PK EV transcript assay may also identify patients who can benefit from a DNA-PK inhibition therapy.