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

Abstract

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.

Circulating Biomarkers for Cancer Detection: Could Salivary microRNAs Be an Opportunity for Ovarian Cancer Diagnostics?

“…The search for miRNA biomarkers is advancing, although, to date, none has yet reached clinical applications [ 81 ]. Papers dealing with salivary miRNAs BMs for PC [ 75 , 76 , 77 , 82 , 83 ], CRC [ 78 , 84 ], LiC [ 79 , 85 ], LC [ 86 ], BC [ 87 ], GC [ 88 , 89 ], and PrC [ 90 , 91 ] are summarized in Table 1 . MiRNAs were detected both as cell free or as exosomal miRNAs, both in saliva alone as well as in other parallel biofluids.…”

Non-Invasive Biomarkers for Immunotherapy in Patients with Hepatocellular Carcinoma: Current Knowledge and Future Perspectives

“…Nevertheless, further studies based on human FMT are needed to confirm its efficacy as a co-adjuvant treatment for a successful immunotherapy. The influence of diet, ethnicity, lifestyle, and chronic therapies on the relationship between gut microbiota and the host [ 212 , 213 , 214 ] are other data that need to be taken into account to further stratify patients and refine gut microbiota’s prognostic significance and therapeutic usefulness.…”

“…Nowadays, liver biopsy is not mandatory for the diagnosis of HCC in patients with liver cirrhosis and is mainly performed for the enrollment of patients in clinical trials; the liquid biopsy analysis of circulating tumor biproducts has emerged as a simple and reproducible tool for monitoring cancer progression and assessing pharmacological efficacy, with the possibility of multiple re-evaluations during treatments [ 23 , 117 ]. Studies aimed at identifying noninvasive biomarkers in easy-to-analyze body fluids such as saliva are being continuously published, but interesting results aside, the generalizability is limited by the small cohorts of patients [ 214 , 215 ]. Unraveling the characteristics of HCC and its response to ICIs through the analysis of samples derived from body fluids is fascinating.…”

Improving the Diagnostic Potential of Extracellular miRNAs Coupled to Multiomics Data by Exploiting the Power of Artificial Intelligence

“…Salivary miRNAs obtained by small RNA sequencing have been validated using ML with random forests as accessible and affordable biomarkers of alcohol dependence (AD) in a large group of 56 African-Americans and 64 European-Americans ( Rosato et al, 2019 ). In another study, by applying support vector machine (SVM) models and leave-one-out cross-validation, the authors reported that salivary miRNAs were effective non-invasive biomarkers of hepatocellular carcinoma ( Mariam et al, 2022 ).…”