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©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
Comprehensive evaluation of microRNA as a biomarker for the diagnosis of hepatocellular carcinoma
Juliane Malik, Martin Klammer, Vinzent Rolny, Henry Lik-Yuen Chan, Teerha Piratvisuth, Tawesak Tanwandee, Satawat Thongsawat, Wattana Sukeepaisarnjaroen, Juan Ignacio Esteban, Marta Bes, Bruno Köhler, Magdalena Swiatek-de Lange
Juliane Malik, Martin Klammer, Vinzent Rolny, Magdalena Swiatek-de Lange, Roche Diagnostics GmbH, Penzberg 82377, Germany
Henry Lik-Yuen Chan, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong 999077, China
Teerha Piratvisuth, NKC Institute of Gastroenterology and Hepatology, Songklanagarind Hospital, Hat Yai 90112, Thailand
Tawesak Tanwandee, Division of Gastroenterology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
Satawat Thongsawat, Department of Internal Medicine, Maharaj Nakorn Chiang Mai Hospital, Chiang Mai University, Chiang Mai 50200, Thailand
Wattana Sukeepaisarnjaroen, Faculty of Medicine, Srinagarind Hospital, Khon Kaen University, Khon Kaen 40000, Thailand
Juan Ignacio Esteban, Liver Unit, Vall d’Hebron University Hospital, Barcelona 08035, Spain
Marta Bes, Transfusion Safety Laboratory, Banc de Sang i Teixits, Barcelona 08005, Spain
Bruno Köhler, Department of Medical Oncology, National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg 69120, Germany
Bruno Köhler, Liver Cancer Center Heidelberg, University Hospital Heidelberg, Heidelberg 69120, Germany
Author contributions: Swiatek-de Lange M provided study supervision; Malik J and Swiatek-de Lange M contributed to project development; Chan HL-Y, Piratvisuth T, Tanwandee T, Thongsawat S, Sukeepaisarnjaroen W, Esteban JI, Bes M and Köhler B contributed to sample collection; Malik J contributed to development of methodology and collection of data; Malik J, Klammer M and Rolny V contributed to the biostatistical analysis of the data; Malik J, Klammer M, Rolny V and Swiatek-de Lange M contributed to the interpretation of the data; Malik J, Klammer M and Swiatek-de Lange wrote the manuscript; Malik J and Swiatek-de Lange M provided critical review and editing of the manuscript; All authors have read and approved the final manuscript.
Institutional review board statement: The study was approved by the following review boards: Siriraj Institutional Review Board; Research Ethics Committee of the Faculty of Medicine, Chiang Mai University; Joint Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee; Ethikkommission Medizinische Fakultät Heidelberg; Research Ethics Committee, Faculty of Medicine, Prince of Songkla University; Clinical Research Ethics Committee of Vall d’Hebron University Hospital; and Khon Kaen University Ethics Committee for Human Research.
Informed consent statement: Written informed consent was obtained from all participants.
Conflict-of-interest statement: Malik J, Klammer M, Rolny V and Swiatek-de Lange M are employees of Roche Diagnostics GmbH and shareholders in Roche Diagnostics.
Data sharing statement: No additional data are available.
STROBE statement: The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
https://creativecommons.org/Licenses/by-nc/4.0/ Corresponding author: Juliane Malik, PhD, Roche Diagnostics GmbH, Nonnenwald 2, Penzberg 82377, Germany.
juliane.malik@roche.com
Received: January 28, 2022
Peer-review started: January 28, 2022
First decision: April 10, 2022
Revised: May 20, 2022
Accepted: July 11, 2022
Article in press: July 11, 2022
Published online: August 7, 2022
Processing time: 186 Days and 20.6 Hours
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
miRNAs alone or as a signature in combination with protein biomarkers AFP and PIVKA-II do not improve the diagnostic performance of the protein biomarkers.
Core Tip: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and has a high mortality rate, making early diagnosis essential for better treatment outcomes. Studies have shown microRNAs to be early markers for HCC; we evaluated the potential diagnostic role of microRNAs alone and in combination with known protein biomarkers (alpha-fetoprotein, protein induced by vitamin K absence/antagonist-II) in samples from HCC-affected individuals and controls using real-time quantitative PCR and next-generation sequencing. MiR-21 and miR-423 demonstrated significant differential expression between the HCC and control groups. MicroRNAs alone or as a signature in combination with protein biomarkers did not improve diagnostic performance of the protein biomarkers.
