Liver cancer is one of the most lethal cancers worldwide. It has a highly aggressive disease course, with a 5-year survival rate of only 18%. When discovered in the early stages, surgery with curative intent is possible.

However, there is a recurrence rate of around 70%. Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer and accounts for up to 90% of all cases.

HCC typically develops in a damaged liver, characterized by the presence of inflammation, fibrosis, and cirrhosis. Chronic liver injury leading to HCC is mainly caused by chronic hepatitis due to infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), long-lasting alcohol abuse, and non-alcoholic fatty liver disease. HCC risk factors include diabetes, obesity, chronic HBV or HCV infection, exposure to dietary aflatoxin, alcohol-induced cirrhosis, and smoking.

HCC is resistant to conventional cancer treatments, such as chemotherapy. HCC is more responsive to targeted therapies, such as kinase inhibitors, immune checkpoint inhibitors, and anti-angiogenesis antibody treatment.

Curative treatments include liver resection, tumor ablation, and liver transplantation. Although liver resection has a 5-year survival rate of 70%, it unfortunately also has a recurrence rate of 70-80%. A liver transplant has a better survival rate and only a 15% recurrence rate. However, insufficient organs are available, and patients are selected for this procedure.

Life-prolonging treatments include transarterial chemoembolization, radiation, and systemic therapy. First-line systemic therapy in the advanced stage includes multi-tyrosine kinase inhibitors, such as sorafenib and lenvatinib.

Additionally, a combinatorial regimen has been FDA-approved, consisting of the immune checkpoint inhibitor, atezolizumab, and the angiogenesis inhibitor, bevacizumab. Second-line treatment includes a sorafenib analog (regorafenib), and immune checkpoint inhibitors, either as single agents (nivolumab) or in combination (ipilimumab and pembrolizumab).

Advances in scientific methods, such as the advent and increased use of NGS, have greatly improved our knowledge of HCC, and the aberrant epigenetic and genetic landscape associated with and contributing to carcinogenesis. This has also enabled the classification of HCCs into different molecular subgroups, associated with different biological and clinical characteristics.

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DNA Methylation in hepatocellular carcinoma

Currently, the most common tools for the diagnosis of liver cancer include ultrasound imaging and alpha-fetoprotein (AFP) serum levels. However, the imaging is limited by interoperator variability and low resolution, and the AFP serum test has a hepatocellular carcinoma (HCC) diagnosis sensitivity of around 60%. AFP is also raised in other liver conditions, and so is not specific to liver carcinoma.

Additionally, about 30% of early-stage HCC cannot be detected using AFP, causing diagnosis and treatment to be delayed. Therefore, there is an urgent need to discover new biomarkers for liver cancer screening, early diagnosis, and prognosis.

DNA methylation is an epigenetic mechanism that affects the gene expression profile of the cell, without altering the genomic sequence. DNA methylation commonly consists of the addition of a methyl group to cytosines in CpG dinucleotides. CpG dinucleotides are non-randomly distributed in the DNA. CpG-islands (CGIs) are CpG-rich regions, between 200bp and several kilobases in length, and take up 1-2% of the genome.

CGIs are typically located in relation to gene promoter and enhancer regions, and to transcription start sites. Methylated CGIs are associated with long-term transcriptional repression. A family of DNA methyltransferases (DNMTs) tightly regulates the methylation pattern. DNMT1 maintains the existing methylation pattern during replication, and DNMT3a and DNMT3b perform de novo methylation during development.

Aberrant DNA methylation levels are generally a hallmark of cancer. In HCC, dysregulated DNA methylation is an early event in cancer initiation, leading to genome-wide hypomethylation of CpG dinucleotides and hypermethylation of CGIs.

Known tumor suppressor genes, including p16, RASSF1A, RUNX3, and DLC-1, are differentially methylated in HCC, resulting in the downregulation of their expression. Several studies have examined the presence of aberrantly methylated genes in HCC, and have demonstrated methylation dysregulation in multiple genes compared to adjacent normal tissue.

Shen et al. investigated a panel of methylated genes in tumor tissue and showed that tumor-specific aberrant methylation could be found in HCC patient plasma as well.

In 87% of the HCC patients, at least one gene of the five in the panel could be identified as hypermethylated in plasma, demonstrating the potential for methylated genes as early biomarkers of HCC.

Drugs targeting epigenetic regulation are a new avenue for HCC treatment research. These drugs can target the DNMT family to alter the genome-wide methylation patterns. DNMT inhibitors are mainly divided into nucleoside inhibitors and nonnucleoside inhibitors.

The nucleoside inhibitor group consists of nucleoside analogs that trap the DNMTs and includes the drugs 5-Azacytidine, Zebularine, and Guadecitabine (SGI-110).

Although these drugs all have an anticancer effect, they have unfavorable side effect profiles and their selectivity towards cancer cells has been low in clinical trials, and further research is needed.

Nonnucleoside inhibitors are being developed as a less toxic alternative to nucleoside analogs. The nonnucleoside inhibitor Doxorubicin is an anthracycline topoisomerase inhibitor. It is associated with high toxicity if administrated conventionally.

However, new promising research has shown a more favorable side effect profile if administered through drug-eluting bead transarterial embolization. Another nonnucleoside inhibitor is SGI-1027, a quinoline-based small-molecule inhibitor of DNMT1, -3A, and -3B. Although the mechanism is not completely clear, SGI-1027 decreases CpG-island hypermethylation, induces DNMT1 degradation, and reverses the epigenetic silencing of tumor suppressor genes, including certain proapoptotic genes (such as BCL-2 and BAX).

The main limitation of nonnucleoside inhibitors is the side effect at the effective dose.
A promising area of study is the combination of epigenetic drugs with traditional anti-HCC drugs. This has the potential to both improve efficacy and reduce side effects. For example, both the combination of low-dose SGI-110 and oxaliplatin or doxorubicin with sorafenib holds great potential in the treatment of liver carcinoma.

A great limitation in the treatment of advanced liver carcinoma is the development of therapy resistance. Drug resistance develops based on multiple mechanisms, involving genetic and epigenetic alterations, epithelial-to-mesenchymal transition, and cancer stem cells.

An important resistance mechanism in HCC is the hypomethylation of the ATP-binding cassette transport proteins. These proteins are ATP-dependent drug efflux pumps that can transport drugs, such as sorafenib, out of the cell. Hypomethylation increases the expression of drug efflux pumps, causing a decrease in drug concentration within the cell and weakening the cytotoxicity of the drug.

Hypermethylation of specific genes involved in the DNA damage response can lead to repressed DNA repair, genomic instability, higher mutation rate, and drug resistance.

Abnormal methylation also occurs at the level of the whole genome. Altered genomic methylation levels can lead to genomic instability and the development of drug resistance, although the mechanism behind this has not yet been elucidated.

Increased whole genome methylation potentially causes tumor cell drug resistance through selective silencing of genes involved in DNA damage repair, apoptosis, and cell cycle signaling.

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How MethylDetect can assist you in your research

At MethylDetect, we can provide you with ready-to-use kits for DNA methylation analysis of your target of interest. In our catalog, we offer more than 850 EpiMelt assays. In Products, you will find EpiMelt kits targeting genes relevant for hepatocellular carcinoma research, such as p16, RASSF1A, and RUNX3.

The EpiMelt assay kits are based on the Methylation-Sensitive High-Resolution Melting (MS-HRM) technology and can be used with standard laboratory equipment for qPCR and melting assessment. Each EpiMelt assay kit comes with a unique control system, securing high sensitivity.

Please consult our catalog at Products, and the protocol at Assay Protocol MethylDetect, for further information on setting up the EpiMelt analysis in your laboratory.

Custom-Tailored EpiMelt Kits

If your target gene is not found in our portfolio, we offer to design and produce EpiMelt assay kits tailored to target specific areas of the genome.

Following methylation-specific array screening analyses, you may have identified targets, which are not yet described in the literature. In collaboration with you, we can design and produce EpiMelt assay kits targeting these specific genomic areas, and tailor the kit to fulfill your needs.

We take into account if your samples are FFPE tissue, liquid biopsies, or high-quality DNA. Customer-tailored EpiMelt assays are always performed in close collaboration with you.

For further information, please contact us at info@methyldetect.com 

Further reading

Fernández-Barrena, M. G., et al. (2020). Epigenetics in hepatocellular carcinoma development and therapy: The tip of the iceberg. JHEP Rep, 2(6), 100167.

Liu, A., et al. (2020). A novel strategy for the diagnosis, prognosis, treatment, and chemoresistance of hepatocellular carcinoma: DNA methylation. Med Res Rev, 40(5), 1973-2018.

Luo, P., et al. (2020). Current Status and Perspective Biomarkers in AFP Negative HCC: Towards Screening for and Diagnosing Hepatocellular Carcinoma at an Earlier Stage. Pathol Oncol Res, 26(2), 599-603.

Shen, J., et al. (2012). Genome-wide DNA methylation profiles in hepatocellular carcinoma. Hepatology, 55(6), 1799-1808.

Toh, T. B., et al. (2019). Epigenetics of hepatocellular carcinoma. Clin Transl Med, 8(1), 13.

Vogel, A., et al. (2022). Hepatocellular carcinoma. Lancet, 400(10360), 1345-1362.

Wu, X., et al. (2020). Circulating tumor DNA as an emerging liquid biopsy biomarker for early diagnosis and therapeutic monitoring in hepatocellular carcinoma. Int J Biol Sci, 16(9), 1551-1562.