Comprehensive evaluation of microRNA as a biomarker for the diagnosis of hepatocellular carcinoma

Abstract

BACKGROUND Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Current guidelines for HCC management recommend surveillance of high-risk patients every 6 mo using ultrasonography. Serum biomarkers, like alpha-fetoprotein (AFP), protein induced by vitamin K absence/antagonist-II (PIVKA-II) and lectin-reactive AFP, show suboptimal performance for detection of HCC, which is crucial for successful resection or treatment. Thus, there is a significant need for new biomarkers to aid early diagnosis of HCC. Studies have shown that the expression level of human microRNAs (miRNAs), a small, non-coding RNA species released into the blood, can serve as an early marker for various diseases, including HCC. AIM To evaluate the diagnostic role of miRNAs in HCC as single markers, signatures or in combination with known protein biomarkers. METHODS Our prospective, multicenter, case-control study recruited 660 participants (354 controls with chronic liver disease and 306 participants with HCC) and employed a strategy of initial screening by two independent methods, real-time quantitative PCR ( n = 60) and next-generation sequencing ( n = 100), to assess a large number of miRNAs. The results from the next-generation sequencing and real-time quantitative PCR screening approaches were then combined to select 26 miRNAs (including two putative novel miRNAs). Those miRNAs were analyzed for their diagnostic potential as single markers or in combination with other miRNAs or established protein biomarkers AFP and PIVKA-II via real-time quantitative PCR in training ( n = 200) and validation cohorts ( n = 300). RESULTS We identified 26 miRNAs that differentiated chronic liver disease controls from (early) HCC via two independent discovery approaches. Three miRNAs, miR-21-5p (miR-21), miR-320a and miR-186-5p, were selected by both methods. In the training cohort, only miR-21, miR-320d and miR-423 could significantly distinguish ( Q < 0.05) between the HCC and chronic liver disease control groups. In the multivariate setting, miR-21 with PIVKA-II was selected as the best combination, resulting in an area under the curve of 0.87 for diagnosis and area under the curve of 0.74 for early diagnosis of HCC. In the validation cohort, only miR-21 and miR-423 could be confirmed as potential HCC biomarkers. A combination of miRNAs did not perform better than any single miRNA. Improvement of PIVKA-II performance through combination with miRNAs could not be confirmed in the validation panel. Two putative miRs, put-miR-6 and put-miR-99, were tested in the training and validation panels, but their expression could only be detected in very few samples and at a low level (cycle threshold between 31.24 and 34.97). CONCLUSION ...

MicroRNAs as Plasma Biomarkers of Hepatocellular Carcinoma in Patients with Liver Cirrhosis—A Cross-Sectional Study

“…For instance, Fang et al [51] suggested that a combination of miR-16, miR-122 and the protein alpha-fetoprotein could increase AUC to 0.862. On the other hand, and in concordance with our results, Malik et al [28] showed that adding miRNAs to the protein biomarkers AFP or DCP did not improve the diagnostic performance further. These results reflect the diverging roles of miRNAs in the diagnosis of HCC, indicating that further research is needed.…”

“…Furthermore, many miRNAs may be both up-and down-regulated in HCC patients, which could lead to analytical difficulties in clinical practice. However, a small number are reported to be upregulated in HCC patients in more than a few publications, the most common of which are miR-21 [11,15,23,[26][27][28], miR-29 [22,26,[29][30][31], miR-192 [11,15,30], miR-199 [15,31,32], miR-221 [26,33,34], miR-222 [26,34], and miR-224 [18,32,35]. Of four meta-analyses that evaluated the diagnostic value of miRNAs for HCC detection, three cautiously encouraged their role as biomarkers [27,36,37], whereas one concluded that adding miRNA to the protein biomarkers AFP or DCP did not improve the diagnostic performance [28].…”

Diagnostic Significance of hsa-miR-21-5p, hsa-miR-192-5p, hsa-miR-155-5p, hsa-miR-199a-5p Panel and Ratios in Hepatocellular Carcinoma on Top of Liver Cirrhosis in HCV-Infected Patients

“…Blood hsa-miRs are of interest for the research of illness prognosis and the detection of cancer due to their great stability in blood and other liquid biopsy samples [ 38 ]. In the current study, combining each of the investigated miRs individually to AFP ROC curve analysis yielded an improved AUC each time compared to either alone.…”

“…During the ROC curve analysis, we decided to increase AFP cutoff to 23.3 ng/mL to ensure the inclusion of AFP-negative HCC cases (12/39) [ 32 ]. However, Malik et al [ 38 ] stated that hsa-miR-21-5p alone or in combination with AFP did not improve the diagnostic performance of the protein biomarkers. Moreover, we thought to investigate miRs’ fold change ratios.…”

Circulating miRNA’s biomarkers for early detection of hepatocellular carcinoma in Egyptian patients based on machine learning algorithms

“…Oncogenes and tumour suppressor genes are both dysregulated during the complicated aetiology of HCC [35][36][37] . By controlling tumour suppressor genes, miRNAs have been shown to be crucial in the development of tumours 38 .…”