Abstract
Hepatocellular carcinoma (HCC) is the most common liver cancer and second leading cause of cancer related death worldwide. Most HCCs occur in a damaged cirrhotic background and it may be difficult to discriminate between regenerative nodules and early HCCs. No dependable molecular biomarker exists for the early detection of HCC. MicroRNAs (miRNAs) have attracted attention as potential blood-based biomarkers. To identify circulating miRNAs with diagnostic potential in HCC, we performed preliminary RNAseq studies on plasma samples from a small set of HCC patients, cirrhotic patients and healthy controls. Then, out of the identified miRNAs, we investigated miR-101-3p, miR-106b-3p, miR-1246 and miR-411-5p in plasma of independent HCC patients’ cohorts. The use of droplet digital PCR (ddPCR) confirmed the aberrant levels of these miRNAs. The diagnostic performances of each miRNA and their combinations were measured using Receiver Operating Characteristic (ROC) curve analyses: a classifier consisting of miR-101-3p, miR-1246 and miR-106b-3p produced the best diagnostic precision in plasma of HCC vs. cirrhotic patients (AUC = 0.99). A similar performance was found when the levels of miRNAs of HCC patients were compared to healthy controls (AUC = 1.00). We extended the analyses of the same miRNAs to serum samples. In serum of HCC vs. cirrhotic patients, the combination of miR-101-3p and miR-106b-3p exhibited the best diagnostic accuracy with an AUC = 0.96. Thus, circulating miR-101-3p, miR-106b-3p and miR-1246, either individually or in combination, exhibit a considerable potential value as diagnostic biomarkers of HCC.
Highlights
Hepatocellular carcinoma (HCC) ranks fifth in incidence and second for cancer mortality worldwide, with nearly 746,000 deaths in 2012 [1]
In addition to hepatitis B and C viral infections and alcohol abuse as etiologic factors, cirrhosis may develop in nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)
After an initial RNAseq discovery phase performed on a small number of plasma samples, 4 miRNAs were further analyzed in independent cohorts of patients and controls to evaluate their significance as diagnostic circulating biomarkers using a droplet digital PCR (ddPCR) approach [33]. ddPCR emerged as a robust method to quantify circulating miRNAs [29,30,31]; compared to real time PCR, ddPCR shows lower variations, it can better reach a statistically significant discrimination between cases and controls [30]
Summary
Hepatocellular carcinoma (HCC) ranks fifth in incidence and second for cancer mortality worldwide, with nearly 746,000 deaths in 2012 (according to the International Agency for Research on Cancer, IARC) [1]. Cirrhosis is present in 80–90% of HCC patients and represents the main risk factor for liver cancer [2]. Survival drops to 3% in advanced HCCs [5], highlighting the need of diagnostic biomarkers for the early detection of HCC in at-risk subjects. Current diagnostic methods such as imaging techniques and serological tumor markers are far from optimal. Accuracy of commonly used blood markers, alpha-fetoprotein (AFP) or des-gamma-carboxy prothrombin is modest, with false negative results in one-third of cases of HCC and a high rate of false positive results in patients with benign liver diseases, such as hepatitis and cirrhosis [7,8,9]. The American Association for the Study of Liver Diseases guidelines (AASLD) no longer recommends AFP to be used for diagnosis [10]
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