Lung cancer is the leading cause of cancer related deaths world wide. Often patients with lung cancer are diagnosed when the cancer is in an advanced stage with metastatic spread locally or to distant organs, resulting in a poor prognosis for the patient. The 5-year survival rate of lung cancer patients depends on the disease stage at time of diagnosis, but is in the range of 4-17%. Smoking is the major risk factor, with 85% of the cases being attributed to carcinogens from cigarette smoking.
Lung cancer is divided into two major groups: small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC comprise approximately 15% of all lung cancer cases, it is associated with tobacco smoking, and is considered the most aggressive form for lung cancer with a two-year survival of only 10%. Most patients are diagnosed with lung adenocarcinoma (AdC) a subtype of NSCLC, which also constitutes of squamous cell carcinoma (SCC), large-cell carcinoma (LCC), and some rare subtypes. Molecular targeted therapy has advanced during recent years due to development of high-throughput sequencing technologies, making it accessible as a diagnostic tool for daily use in the clinic. Focus here is to block activated kinases in different pathways involving targets as: EGFR, KRAS, BRAF, ALK, and c-MET. TP53, a tumor suppressor gene, is found mutated in more than 90% of the tumors especially in advanced lung cancer. Mutations and other genetic alterations have been identified in lung cancer, which is able to silence gene expression, but lately focus has turned to epigenetic alterations affecting the tumor epigenome, as a key to understand the aetiology of lung cancer.
DNA methylation lung cancer
Epigenetic alterations affecting gene expression is represented by an interplay between histone modifications and DNA methylation levels, to either provide an open chromatin structure, accessible for the transcriptional machinery, or a closed conformation silencing the affected genes. DNA methylation has been widely studied in cancer, and analysis of DNA methylation alterations in lung cancer has identified a number of differentially methylated regions affecting specific genes involved in pathways defined as hallmarks of cancer. (For background information on the mechanisms behind DNA methylation, please see the section DNA methylation and cancer at: https://www.methyldetect.com/dna-methylation-in-cancer/).
In brief, DNA methyltransferases mediates the transfer of methyl groups to the 5th carbon in cytosine, when the cytosine is part of a CpG dinucleotide. These CpG sites tend to cluster in promoters and other regulatory regions of the genome forming the so called CpG islands. DNA methylation affecting a CpG island may cause transcriptional silencing, whereas methylation of CpG sites in a gene body may result in an increased transcription of the gene. Studies comparing the DNA methylation patterns of normal lung tissue to lung cancer cell DNA have identified a number of genes affected by aberrant promoter hypermethylation, in which the methylation status is significantly associated with prognosis or being a diagnostic marker for the individual lung cancer patient.
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DNA methylation biomarkers
Aberrant DNA methylation has affected a number of genes in lung cancer cells. Especially DNA hypermethylation has silenced genes and thereby impaired important functions as: DNA repair ( RASSF1A and MGMT), apoptosis (DAPK, P14, OTUD4), cell cycle (CDKN2A/p16), and metastasis (RARβ, CDH1/E-cadherin). Promoter methylation of the adenomatous polyposis coli (APC) gene, which frequently is found mutated in colorectal cancer, may eventually result in abnormal activation of the Wnt/β-catenin signalling pathway, affecting important cellular functions as: cell proliferation, cell polarity and tissue homeostasis. Aberrant promoter methylation of the RUNX3 gene leads to deregulation of the TGF-β signalling pathway, thereby promoting tumorigenesis. Epigenetic silencing of genes involved in maintaining important pathways to maintain normal cell growth, the hallmarks of cancer, can play a role as biomarkers for early diagnosis (as CDKN2a/p16 for non-small cell lung cancer and RASSF1A), or prognosis (RASSF1A, OTUD4).
To obtain a high specificity of the DNA methylation markers, focus has been directed towards establishing a panel of differentially methylated CpG sites to act as prognostic, predictive or early detecting biomarkers. By comparing the methylation pattern between lung adenocarcinoma and adjacent normal lung tissue of four loci: hypermethylation of HOXA9, hypomethylation of KRTAP8, CCND1 and TULP2 showed the potential as a panel of markers for early diagnosis of lung adenocarcinoma (Shen N. et al. 2019).
Hypermethylation of MGMT is a well-known predictive marker for treatment response to alkylating chemotherapy and ionising radiation both in glioblastoma and colorectal cancer. To explore the possibility to use epigenetic -based biomarkers to predict drug response for lung cancer patients, a study on small-cell lung cancer cell lines, in which gene expression, methylation and drug response data from the cell line were available, identified a number of potential targets (see Krushkal et al. 2020). Among these, TREX1 methylation was found to be associated with SCLC sensitivity to Aurora kinase inhibitors, antimitotic agents, a CDK inhibitor, and an ATR inhibitor. EPAS1 methylation was associated to Aurora Kinase inhibitor response, PLK-1 and Bcl-2 inhibitors. SLFN11 methylation predicted drug response to DNA damaging agents and YAP1 to rapamycin.
Detecting aberrant promoter methylation has proved a valuable way to identify new biomarkers available for early diagnosis, prognosis or as predictive markers for the different types of lung cancer. Methylation marks are stable and together with highly sensitive detection technologies the epigenetic-based biomarkers can be assessed both in liquid biopsies as circulating tumor DNA (ctDNA) or in exhaled breath condensate (EBC), which points to a future hope for early detection of the disease.
How MethylDetect can assist your research in lung cancer
MethylDetect provides ready-to-use kits for DNA methylation analysis of your target of interest. On www.MethylDetect.com you will find EpiMelt kits targeting tumor suppressor genes relevant for lung cancer research as: CDKN2A (p16), RASSF1A, CDH1, MGMT, DAPK, APC, RARβ, RUNX3, OTUD4 together with more than 840 other gene-specific assays.
All EpiMelt kits contains a primer mix and a control set of 100% methylated, unmethylated, and an assay calibration control, to ensure the high sensitivity of your EpiMelt assays. Standard qPCR platforms with a high-resolution melting module can be used, please consult the protocol at https://www.methyldetect.com/methyldetect-kit-for-dna-methylation-assessment/, for further information on setting up the EpiMelt analysis in you laboratory.
If your target of interest is missing from our portfolio, we design and produce EpiMelt kits in collaboration with you, to ensure that the design fits your requirements for test material and expected methylation status (hypo- or hypermethylation). We design EpiMelt kits for DNA derived from sources as: FFPE tissue, liquid biopsies or high quality DNA.
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Shi Y-X et al. Current Landscape of Epigenetics in Lung Cancer: Focus on the Mechanism and Application. Journal of Oncology. Volume 2019, Article ID 8107318,
Nunes SP et al. Subtyping Lung Cancer Using DNA Methylation in Liquid Biopsies. J. Clin. Med. 2019, 8, 1500
Shen N et al. A Diagnostic Panel of DNA Methylation Biomarkers for Lung Adenocarcinoma. Frontiers in Oncology, December 2019 | Volume 9 | Article 1281
Krushkal J et al. Epigenome-wide DNA methylation analysis of small cell lung cancer cell lines suggests potential chemotherapy targets. Clinical Epigenetics (2020) 12:93.
The cancer genome atlas (TCGA): https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga