Page 157 - D. Cancer biology

Application of methylation sensitive high-resolution melting
analysis for prediction of prognosis in liver cancer patients

Si-Cho Kim , Dongwoon Han , Jeongsil Ha and Young-Joon Kim 1,2*
3
3
1
1 Department of Integrated Omics and Biomedical Science, Yonsei University, Seoul 03722, Korea
2 Department of Biochemistry, Yonsei University, Seoul 03722, Korea
3 Department of Integrative Bioscience & Biotechnology, Sejong University, Seoul 05006, Korea
Abstract

Primary liver cancer is the sixth most common cancer and the second leading cause of cancer-related mortality. Most histologic
types of liver cancer are hepatocellular carcinoma (HCC), accounting for approximately 80% of liver cancer patients. As a cause of
HCC, various risk factor such as cirrhosis, obesity, viral hepatitis, age, gender and environmental factor exist and the tendency of
concentration in East Asia and China been confirmed. If liver cancer recurs after surgery, it has been found that the patient survival
rate is relatively deteriorated. Follow-up and appropriate management of prognosis will help improve the patient survival rate. We
conducted a study to predict the prognosis of HCC patients by applying methylation sensitive high-resolution melting (MS-HRM)
analysis based on the prognostic markers found in previous study. As a result, based on the biomarkers we discovered, MS-HRM
analysis could classify each HCC patient according to recurrence. Moreover, the MS-HRM analysis can perform the inspection in an
efficient time and cost compared to qMSP and NGS methods and we intend to conduct research related to the mechanisms
associated with early recurrence of HCC by monitoring many HCC patients through MS-HRM analysis results.

Introduction
Studies that various disease can be determined based on changes
in methylation in CpG present in human genome are being actively
conducted, and particularly in cancer, it can be used as a powerful
diagnostic and prognostic marker. The discovery of several
methylation marker has promoted the development of various
methods for identifying markers, and among them, the most Figure 2. Melt curve and
widely used method is a PCR-based method. For PCR-based Difference RFU for
synthesized oligonucleotide.
methylation confirmation, there are methylation-specific PCR(MSP) The difference RFU value is
and qMSP that specifically confirm methylated or unmethylated the RFU of each sample
minus the RFU of the
template, but also methylation independent PCR(MIP) in which completely unmethylated
amplification proceeds regardless of methylation status. MS-HRM, amplicon
one of the MIP methods, is analyzed by measuring melting It was confirmed that the melt curve pattern was clearly divided in
properties of methylated and unmethylated PCR amplicon. (Fig. 1) all patients except on patient with recurrence. Additionally,
We studied a method to predict the prognosis of HCC patients experiments on commercially available human DNA (EpiTect,
using the MS-HRM and confirmed that the patient’s clinical data Qiagen) and cell lines were also confirmed to accurately describe
and experimental results were consistent. the methylation characteristics of each samples.

Figure 3. Melt curve and difference RFU for HCC tissue, cell line and control DNA
Conclusion
Figure 1. Overall schematic diagram of MS-HRM analysis. Numerous methods have been developed to confirm the
Results methylation status of genomic regions of interest, such as absolute
First, we conducted an experiment to confirm the difference in the melt DNA methylation assay and relative DNA methylation assay, but
curve pattern according to the methylation ratio in two different currently, there are very few assays that can be practically used in
synthesized oligonucleotide with completely methylated or unmethylated
template. (Fig. 2) In figure 2, it was confirmed that the melt curve pattern clinical practice. We confirmed that through MS-HRM assay, it is
of completely methylated and unmethylated oligonucleotide was clearly possible to predict the prognosis of HCC patients in an efficient time
distinguished, and as the methylation ratio increased, the Tm of the and cost without using dye-coupled probe or any sequencing
amplicon increased. Second, to demonstrate the utility of our MS-HRM equipment. In the future, we plan to confirm how effectively the
analysis for detecting different patterns of methylation in HCC tissue prognosis can be predicted through the application of assays to many
sample, we conducted an MS-HRM analysis 12 HCC tissue samples HCC patient and various mechanisms related to early recurrence of
included 6 recurrence and 6 non-recurrence. (Fig. 3) HCC will be studied through continuous patient monitoring.

Reference Acknowledgements
1. Wojdacz, Tomasz K., Alexander Dobrovic, and Lise Lotte Hansen. "Methylation-sensitive high- This research was supported by Collaborative Genome Program
resolution melting." Nature protocols 3.12 (2008): 1903. for Fostering New Post-Genome industry through the National
2. Smith, Eric, Michael E. Jones, and Paul A. Drew. "Quantitation of DNA methylation by melt curve
analysis." BMC cancer 9.1 (2009): 123. Research Foundation of Korea(NRF) funded by the Ministry of Si-Cho Kim
3. Hussmann D., Hansen L.L. (2018) Methylation-Sensitive High Resolution Melting (MS-HRM). In: Tost J. Science ICT and Future Planning. Cancer Genome Research Center
Yonsei University, Seoul, S. Korea
(eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. (grant number : 2016M3C9A492172). http://cgrc.Yonsei.ac.kr/g5/
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