Supplementary Material for: DETECTION OF CIRCULATING mRNA VARIANTS IN HEPATOCELLULAR CARCINOMA PATIENTS USING TARGETED RNAseq

Introduction. Mutations in circulating nucleic acids are biomarkers for the early detection and management of hepatocellular carcinoma (HCC). While circulating tumor DNA (ctDNA) and microRNA have been extensively explored circulating tumor mRNA and circulating mRNA mutants (ctmutRNA), which may provide advantages over other analytes, remain less well described. We previously reported the identification of 288 HCC selective ctmutRNA variants, called “candidates,” from a small cohort of HCC patients using total RNAseq. The objective of the current study was to use targeted RNAseq to validate the specificity and sensitivity of these HCC selective variants in an independent cohort of patients with liver cirrhosis (LC). Methods: Several methods to isolate small Extracellular Vesicles (sEVs) and amplify mRNA from the circulation were compared. RNA was isolated, and the primers and probes selective for the 288 regions of interest were used with RNA from HCC (N=50) and LC and no HCC (N=35) patients. HCC tumor tissues (N=11), a normal liver tissue and 3 cell lines were also studied. cDNA synthesis was followed by library construction using QIAseq RNA Fusion XP panel. QC analysis was carried out with an Agilent Bioanalyzer before sequencing on a NextSeq 550 instrument. A GATK HaplotypeCaller was used for variant calling and annotation carried out using snpEff. Results: Among the test panel of 288 ctmutRNA candidates in the original cohort, 75 were detected in the new cohort of plasma samples. Moreover, 388 other variants in proximity to the original lesions were also found in multiple HCC but not LC plasma samples. A subset of 36 HCC selective variants were able to identify all HCC patients. The most common tumor specific variants were Indels and SNPs. Novel mRNA fusion variants, corresponding to SENP7, HYI, SAR1A, RASA2, TUBA transcripts, etc. were identified in HCC and LC patients. Conclusion: Circulating RNA could be a robust analyte for non-invasive early detection of HCC and ctRNA panels could work as powerful tools in the entire spectrum of clinical management.