Salivary miRNAs as non-invasive biomarkers of hepatocellular carcinoma: a pilot study

Background: Improved detection of hepatocellular carcinoma (HCC) is needed, as current detection methods, such as alpha fetoprotein (AFP) and ultrasound, suffer from poor sensitivity. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate many cellular functions and impact cancer development and progression. Notably, miRNAs are detectable in saliva and have shown potential as non-invasive biomarkers for a number of cancers including breast, oral, and lung cancers. Here, we present, to our knowledge, the first report of salivary miRNAs in HCC and compare these findings to patients with cirrhosis, a high-risk cohort for HCC. Methods: We performed small RNA sequencing in 20 patients with HCC and 19 with cirrhosis. Eleven patients with HCC had chronic liver disease, and analyses were performed with these samples combined and stratified by the presence of chronic liver disease. P values were adjusted for multiple comparisons using a false discovery rate (FDR) approach and miRNA with FDR P < 0.05 were considered statistically significant. Differential expression of salivary miRNAs was compared to a previously published report of miRNAs in liver tissue of patients with HCC vs cirrhosis. Support vector machines and leave-one-out cross-validation were performed to determine if salivary miRNAs have predictive potential for detecting HCC. Results: A total of 4,565 precursor and mature miRNAs were detected in saliva and 365 were significantly different between those with HCC compared to cirrhosis (FDR P < 0.05). Interestingly, 283 of these miRNAs were significantly downregulated in patients with HCC. Machine-learning identified a combination of 10 miRNAs and covariates that accurately classified patients with HCC (AUC = 0.87). In addition, we identified three miRNAs that were differentially expressed in HCC saliva samples and in a previously published study of miRNAs in HCC tissue compared to cirrhotic liver tissue. Conclusions: This study demonstrates, for the first time, that miRNAs relevant to HCC are detectable in saliva, that salivary miRNA signatures show potential to be highly sensitive and specific non-invasive biomarkers of HCC, and that additional studies utilizing larger cohorts are needed.