Cancer diagnostic classifiers based on quantitative DNA methylation

Abstract

Epigenetic change is part of the carcinogenic process and a deep reservoir for biomarker discovery. Reversible methylation of cytosines is noteworthy because it can be measured accurately and easily by various molecular methods and DNA methylation patterns are linked to important tumourigenic pathways. Clinically relevant methylation changes are known in common human cancers such as cervix, prostate, breast, colon, bladder, stomach and lung. Differential methylation may have a central role in the development and outcome of most if not all human malignancies. The advent of deep sequencing holds great promise for epigenomics, with bioinformatics tools ready to reveal large numbers of new targets for prognosis and therapeutic intervention. This review focuses on two selected cancers, namely cervix and prostate, which illustrate the more general themes of epigenetic diagnostics in cancer. Also discussed is differential methylation of specific human and viral DNA targets and laboratory methods for measuring methylation biomarkers.

Figure 1.

Examples of DNA methylation modifications on cytosines (C) of CpG dyads and the effects of these changes at the level of the gene and relationship to carcinogenesis. (A) Nucleotide C is shown on the left, this base can be methylated at position 5 of the aromatic ring by one of several DNMT to become 5-methyl cytosine shown on the right. The methylation is reversible and methyl modifications can be changed to hydroxymethyl or other groups or removed by various enzymes. (B) The gross effect of DNA hypermethylation in a gene promoter region, is to turn off transcription of the gene (shown by the curved arrow). Many tumor suppressor genes, such as APC and BCL2 are controlled by methylation, and if the genes are deactivated, then critical checks and balances in cells are removed, which can lead to apoptosis or to carcinogenesis. Similarly, oncogenes such as CCND2 can be activated by removal of methylation marks from important regulatory regions . (C) The methylation levels of a DNA region can be measured by several methods. One of the more comprehensive and convenient methods involves bisulfite conversion, which chemically changes nucleotide C into uracil (U). The U then pairs with adenine (A) and upon replication of DNA by PCR the nucleotide U is converted into thymine (T). This C to T change can be measured by sequencing and forms the basis of many DNA methylation quantitation tests. The affected C and T bases are shown in underlined boldface and a methylated C is shown as C*. The upper strand represents the native DNA sequence and the lower strand the same sequence replicated after PCR amplification.

Figure 2.

DNA methylation patterns across the genome of HPV16, showing the percentage median values for selected CpG sites in cancers (solid circles), cervical intraepthelial neoplasia 2/3 (triangles), and normal women who had transient HPV16 infections (open circles). Other hrHPVs have similar methylation patterns, with relative peaks in the L1 and L2 regions and little to no methylation in the URR. The circular genome is depicted as opened in the URR region. The specific peaks and valleys of the HPV16 genome methylation profile are quite reproducible in specimens from different geographic locations and may reflect the intrinsic relative positions of histones and other binding complexes.