Circulating mRNA variants as a potential biomarker for surveillance of hepatocellular carcinoma

Background and rationale. We previously reported that the liver derived mRNA transcripts are elevated in the circulation of hepatocellular carcinoma (HCC) patients compared to liver cirrhosis (LC) patients without cancer. We are now reporting the detection of high-risk RNA variants exclusively in the circulation of HCC patients potentially offering a novel non-invasive platform for HCC surveillance and early detection. Numerous genomic alleles such as single nucleotide polymorphisms (SNPs), nucleotide insertions and deletions (called Indels), splicing variants in many genes, have been associated with elevated risk of cancer. Approach: RNAseq analyses was carried out in the plasma of 14 individuals with a diagnosis of HCC, 8 with LC and no HCC, and 6 with no liver disease diagnosis. RNA from 6 matching tumors and 5 circulating extracellular vesicle (EV) samples from 14 of those with HCC was also analyzed. HCC specific SNPs and Indels referred as 'variants'were identified using GATK HaplotypeCaller and annotated by SnpEff to filter out high risk variants. Results: The variant calling on all RNA samples enabled the detection of 5.2 million SNPs, 0.91 million insertions and 0.81 million deletions. Comparing tumors, normal liver tissue, plasma and plasma derived EVs by RNAseq we were able to identify 5480 high-risk tumor specific mRNA variants in the circulation of HCC patients. These variants are concurrently detected in tumors and plasma samples or tumors and EVs from HCC patients, but none detected in normal liver, plasma of LC patients or normal healthy individuals. Plasma derived EVs from HCC patients appear to be specifically enriched in a subset of the high-risk variants concordant in tumors. Most of the top tumor specific variants seem to represent splicing variants and SNPs. Apart from representing several cancer pathways, the variants highlight an altered tumor metabolism and point to several potential diagnostic and therapeutic targets. These results demonstrate a high mutation burden in tumors and plasma of HCC patients reflected in mRNA transcripts. The possibility that many of the mRNA transcripts containing SNPs associated with cancer are coming from the liver tumors and possible use in early detection and management is discussed.