DNA Methylation Analysis: a Promising Diagnostic Tool DNA-Methylierungs-Analyse: Ein Vielversprechendes Diagnostisches Werkzeug

J Lab Med 2004;28(3):233–238 2004 by Walter de Gruyter • Berlin • New York 2004/04017

Molekulargenetische Diagnostik Redaktion: H.G. Klein

DNA methylation analysis: a promising diagnostic tool DNA-Methylierungs-Analyse: ein vielversprechendes diagnostisches Werkzeug

Matthias Schuster* sionsmarkern gewidmet ist, etabliert sich die DNA-Me- thylierung mit großer Geschwindigkeit als eine alternative Epigenomics AG, Berlin, Germany zellula¨ re Informationsebene. Die DNA-Methylierung ist an der Regulation der Genaktivita¨ t beteiligt. A¨ nderungen des Abstract Methylierungsmusters werden mit verschiedensten Erkrankungen in Verbindung gebracht. DNA-Methylie- Since the investigation of molecular biological mechan- rungsmarker gelten daher heute zu Recht als vielver- isms was extended to the genomic level and after the sprechende diagnostische Werkzeuge, z. B. fu¨ r die sequence of the human genome was established, inter- Fru¨ herkennung von Krebserkrankungen. Mehrere Me- est in DNA-based diagnostics has increased tremen- thoden stehen fu¨ r die genomweite Entdeckung von Me- dously. While most research is currently being devoted thylierungsmarkern zur Verfu¨ gung, die die Unter- to the discovery and clinical application of genetic, pro- scheidung verschiedener Pha¨ notypen zuverla¨ ssig teomic and RNA expression markers, DNA methylation ermo¨ glichen. Im Gegensatz zu RNA-Expressionsmarkern is rapidly emerging as a new level of cellular information ko¨ nnen DNA-Methylierungsmarker auch in historischen, with biomarker potential. DNA methylation is involved in in Paraffin eingebetteten Geweben gut analysiert werden. the regulation of gene activity. Aberrant DNA methylation Etablierte Methoden erlauben den Nachweis weniger has been associated with a variety of human diseases. DNA-Kopien, die ein spezifisches Methylierungsmuster As such, DNA methylation markers hold a great promise aufweisen, vor dem Hintergrund eines Vielfachen an nor- as a diagnostic tool, e.g. in early cancer screening w1x. mal methylierter DNA. Damit sind alle Voraussetzungen Methods are available for the genome-wide discovery of fu¨ r den Nachweis von DNA-Methylierungsmarkern in methylation markers that differentiate between different Ko¨ rperflu¨ ssigkeiten als gut zuga¨ nglichem klinisch rele- types of tissue samples. In contrast to expression mark- vantem Probenmaterial gegeben. ers, DNA methylation markers are accessible at the DNA level, which enables their analysis even in archived paraffin-embedded tissue samples. Methods are available to Schlu¨ sselwo¨ rter: DNA-Methylierung; Biomarker; mole- detect a few copies of aberrantly methylated DNA in a kulare Diagnostik; Krebs-Screening; Pharmakogenomics. background of a vast excess of normally methylated DNA, which is the prerequisite for assaying methylation DNA methylation markers also in body fluids that are readily accessible in clinical practice. General

Keywords: DNA methylation; biomarker; molecular diag- The genetic information, as it is passed from generation nostics; cancer screening; pharmacogenomics. to generation, is defined by the sequential order of the four nucleobases thymine, adenine, guanine, and cyto- Zusammenfassung sine within a polymeric high molecular desoxynucleic acid. In humans, however, the C-5 methylation of cyto- Seit der Ausweitung der Erforschung molekularbiolo- sine in the sequence context CpG gives rise to 5-meth- gischer Mechanismen auf das genomische Niveau und ylcytosine, which represents a ‘‘fifth’’ base with a unique der erfolgreichen Sequenzierung des Humangenoms ist covalent structure w2x. Cytosine methylation is catalysed das Interesse an DNA-basierten diagnostischen Verfah- by methyltransferases using S-adenosylmethionine as ren enorm gestiegen. Wa¨ hrend die gegenwa¨ rtige For- the methyl donor. The distribution of 5-methylcytosine schung hauptsa¨ chlich der Identifizierung und klinischen throughout the genome is closely related to the transcrip- Anwendung von genetischen, Protein- und RNA-Expres- tional activity of genes. Since 5-methylcytosine exhibits nearly the same base pairing behaviour as cytosine itself, *Correspondence: Matthias Schuster, Epigenomics AG, Kastanienallee 24, 10435 Berlin, Germany proven methods in molecular biology can typically not be E-mail: [email protected] used to understand this interdependence. Indeed, only 234 M. Schuster: DNA methylation analysis

more recent research has highlighted that DNA methyl- affin-embedded tissue, which is extremely beneficial ation is central to a complex framework of ‘‘epigenetic’’ when e.g. considering retrospective clinical studies. The factors that regulate and fine-tune the transcriptional mRNA expression level of genes in one cell differs by activity in most parts of the human genome w3x. These several orders of magnitude. Since the methylation of are factors that bring about alterations in the action of each gene on one allele comprises a binary signal, DNA genes without changing the DNA sequence. It has been methylation signals in mixtures of cells vary between 0% known for some time that the promoters of most human and 100%, i.e. over a linear range. Therefore, the analysis genes contain ‘‘CpG islands’’ that are characterized by of DNA methylation marker panels is technically more an increased density of guanines, cytosines and CpG feasible. In comparison to the above mentioned genomic dinucleotides, and significantly exceed the average classes of biomarkers, changes in the proteome of a cell occurrence within the genome w4x. It is now generally are the closest reflection of its phenotype. Accordingly, accepted that the degree of the CpG methylation of intensive research is devoted to the discovery and eval- these CpG islands can be closely correlated to the chro- uation of proteomic markers w10x, e.g. for cancer screen- matin structure, especially the density of the chromatin ing. However, although significant progress has been w5x. As a rule of thumb, promoters of transcriptionally made in the development of technologies for finding pro- active genes show low methylation levels, whereas tein markers, no method is available for amplifying the promotor hypermethylation leads to a dense chromatin proteome comparable to efficient nucleic acid amplifi- structure that is transcriptionally inactive w6x. According cation methods, such as the polymerase chain reaction to current models, methylated DNA attracts methyl bind- (PCR w11x), enabling systematic approaches for discov- ing proteins that themselves recruit histone deacetylases. ering genomic and epigenomic markers (see below). Histone acetylation is generally associated with active Methylation positions have been identified that corre- chromatin. On the other hand, histone deacetylation, as late with diabetes type II, cancer, atherosclerosis, rheu- associated with DNA methylation, makes the histones matoid arthritis, and disease of the CNS w12x. Methylation accessible to histone methyltransferases. Histone meth- at other positions has been shown to correlate with age, ylation represents inactive chromatin. It is currently not gender, nutrition, drug use, and probably a whole range entirely clear how genes are reactivated, since no of other environmental influences w13x. Due to their enzymes catalyzing the demethylation of histones or potential for molecular diagnostics, our knowledge about DNA have yet been described in sufficient detail. During DNA methylation changes associated with cancer has DNA recombination, the methylation profile of the parent been growing exponentially over the last years and new strand is enzymatically copied onto the complementary markers are reported weekly, if not daily. The emerging strand w7x. This process, which is catalyzed by mainte- pattern of DNA methylation found in cancer cells sup- nance methyltransferases, is the basis for the relative ports two general notions. CpG islands in the promoter stability of the DNA methylation information content and exon 1 regions of tumor suppressor genes have throughout cell divisions and even generations. The inhi- been described to be hypermethylated in a range of dif- bition of this process can lead to gene reactivation. ferent cancers. However, most CpG sites within the human genome are part of repetitive elements that are DNA methylation markers methylated in benign cells. Hypomethylation of these elements in some cancers has been associated with When compared to single nucleotide polymorphisms genomic instability w14x. Although at a lower scale than (SNPs w8x), which are under intensive investigation as hypermethylation, the hypomethylation of gene promot- genetic predisposition markers either alone or in combi- ers in cancer cells has also been described w15x. In com- nation (haplotypes), alterations in DNA methylation can parison to promoter hypermethylation, hypomethylation occur much more frequently. A SNP occurs every 100 to of individual genes currently needs to be considered 300 bases along the 3-billion-base human genome, underexplored, mainly as a result of the limitations of the whereas the number of CpG positions within a CpG analysis methods used today. island is significantly higher. SNPs represent inherited genetic alterations that can potentially be used to describe the likelihood for developing certain diseases. DNA methylation technology They do not normally change over time in an individual. In contrast, DNA methylation is a much more dynamic Marker discovery process. Like its transcriptional profile, the methylation pattern of the DNA of a cell is determined by a whole How do you discover DNA methylation markers differ- spectrum of biochemical parameters w9x. Both phenom- entiating between two types of cells, e.g. benign and ena reflect the phenotype of a cell at a given time. Meth- cancer cells? The methylome of a cell consists of tens ylation markers offer several advantages over expression of thousands of CpG islands associated with individual markers both from a technical and a conceptional point genes and their activity. The number of individual meth- of view. The stability of DNA as the analyte enables methylation positions that could be aberrantly methylated in ylation analysis in difficult samples such as archived par- diseased states is even higher by 3–4 orders of magni- M. Schuster: DNA methylation analysis 235

effort to identify, catalogue and interpret genome-wide DNA methylation patterns of all human genes in all major tissues has been initiated by The Wellcome Trust Sanger Institute (Cambridge, UK), the Centre National de Ge´ no- typage (Paris, France) and Epigenomics AG (Berlin, Ger- many) that have formed a consortium to conduct the Human Epigenome Project (HEP, www.epigenome.org). Based on public versions of the sequence of the human genome established by the Human Genome Project (HUGO w18x) this project will systematically uncover the epigenetic information layer, which – to a large extent – is still hidden. Although not as widely used as sequencing, the methylation specific hybridisation of fluorescently labelled multiplex PCR products onto oligonucleotide Figure 1 Methylation sensitive arbitrarily primed PCR (AP- microarrays as introduced by Epigenomics comprises a PCR): After methylation, sensitive restriction radioactively labelled degenerate primers biased for amplifying CpG rich regions are used to produce a complex amplificate. Differences in the PAGE band pattern (arrows) between samples, which are applied to different lanes, indicate methylation differences. tude. Two groups of methods are amenable to the discovery of methylation markers on a genome-wide scale. The first group relies on the ability of methylation specific restriction enzymes to cleave doublestranded DNA in both a sequence and methylation specific manner w3x.In general, after a clinical question has been defined and appropriate clinical samples for the marker discovery have been identified, DNA is extracted and subjected to restriction by methylation sensitive restriction enzymes, cleaving only when their recognition sequence is unmethylated, leaving completely methylated DNA regions intact. After this step, different techniques are applied to obtain a differential footprint representing the differences of the cleavage pattern between the two samples. During arbitrarily primed PCR (AP-PCR w16x), radioactively labelled degenerate primers biased for amplifying CpG- rich regions are used to produce a complex amplificate that is analysed on polyacrylamide gels (Figure 1). Dif- ferential bands are excised, cloned and mapped against the human genome to identify differentially methylated sequence tags (MeSTs). In other genome-wide MeST discovery methods (MCA, RLGS, DMH), the restriction product is analyzed by utilizing methods such as linker ligation, substractive hybridization, two-dimensional gel electrophoresis or hybridisation to arrayed CpG island libraries. The second group of methods for identifying MeSTs is based on the unique property of inorganic bisulfite to transform unmethylated cytosine to uracil (Figure 2) without affecting 5-methylcytosin w17x. Thus, the bisulfite reaction causes a conversion of the epigenetic methylation information into a (albeit synthetic) true genetic information, the analysis of which benefits from the entire spectrum of molecular genetics techniques, most notably from PCR. A powerful method for analyzing multiple CpG Figure 2 Bisulfite conversion of genomic DNA. (A) Mechanism sites is bisulfite sequencing, which can be performed of the reaction from top to bottom: Sulfonation, Deamination, either directly on PCR products or on cloned PCR prod- Desulfonation; (B) Bisulfite conversion leads to formation of a ucts obtained from bisulfite-treated DNA. A large scale single-stranded product with a new primary structure. 236 M. Schuster: DNA methylation analysis

sufficient DNA in a more homogeneous state. Common assay technology is again based on bisulfite treatment and subsequent PCR. However, in addition to Real-Time PCR, less sensitive methods are also applicable to assess the methylation level of the markers of interest. In general, all techniques that are in routine use for SNP typing can also be applied to this type of methylation analysis, e.g. primer extension, hybridisation of allele specific probes or allele specific ligation w1x.

Clinical applications of DNA methylation markers

Disease detection and monitoring

Early detection of disease, with cancer being the most prominent example, can improve the clinical outcome and thus improve the quality of life or even save lives. Today, the potential of biomarkers for detecting cancer prior to the presentation of first symptoms is generally recognized. However, population-based screening approaches require the detection of marker molecules in Figure 3 Methods for sensitive and specific amplification/ easily accessible body fluids at an early tumor stage. The detection of hypermethylated DNA. (A) Methylation specific PCR availability of techniques for the sensitive detection of (MSP) with methylation specific Taqman probe; (B) Heavy Methyl minute amounts of aberrantly methylated DNA makes (HM) PCR with methylation specific Taqman probe. DNA methylation the ideal parameter for this type of clin- viable and proven method for the analysis of hundreds ical application. Numerous publications describe the of CpG positions in parallel w19x. analysis of methylation markers in samples like serum, plasma and urine from cancer patients in comparison to w x w x DNA methylation assays healthy controls 1 . For example 21 , the HM assay technology has been applied to measure the prevalence Methylation markers with the ability to differentiate of calcitonin promoter hypermethylation in colon cancer between different phenotypes are only of academic inter- patients and healthy donors (Figure 4). Both colon tissue est unless they can be assayed in the relevant tissues. and serum samples were analysed. The methylation sig- Two situations have to be considered. Disease detection nal was normalized for the total amount of DNA, differing will require assays that are able to detect aberrant meth- from sample to sample w22x. The colon tissue assay was ylation of free floating DNA shed from the source organ highly sensitive and specific with an area under the ROC (e.g. a tumor) into body liquids, such as blood or urine. curve (AUC) of 0.84. Out of the six matched cancer Whereas the low analyte concentrations in these samples patient serum samples, five had significantly higher require extremely sensitive assays, the presence of sig- methylation levels (by at least six standard deviations) nificant and varying amounts of background DNA chal- than the mean of the healthy population. This finding lenges the assay specificity. Accordingly, sensitive and demonstrates the suitability of DNA methylation analysis specific methods are a prerequisite for the detection of in blood-based samples for early colon cancer screening. a specific methylation profile in a large background of This type of a diagnostic test has the potential to replace differently methylated DNA. The most common assay tests like the occult blood test that is currently used in applied to sensitive methylation detection today is the routine testing to decide whether colonoscopy should be methylation-specific PCR (MSP w20x) on bisulfite-treated performed or not. DNA. This reaction is a variation of allele-specific PCR, The power of methylation for disease detection is not the specificity of which can be enhanced by performing limited to early detection in body fluids. In a recent pub- it in real-time using methylation specific probes (Figure lication w23x, the utility of GSTP1 promoter methylation 3a). Only recently, the HeavyMethyl (HM w21x) PCR tech- analysis in prostate sextant biopsies to supplement the nology has been reported to be an alternative assay for- histologic review has been demonstrated. According to mat where selective blocking of background DNA is used the current practice, prostate cancer is diagnosed by dig- for methylation specific amplification (Figure 3b). In con- ital rectal examination and the measurement of serum trast to sensitive detection, the molecular classification prostate-specific antigen (PSA) levels, followed by trans- of tissues (e.g. of aggressive vs. nonagressive tumors) rectal prostatic needle biopsy. Biopsy analysis is neces- can often be performed on tissue samples containing sary, since PSA levels can be increased also in benign M. Schuster: DNA methylation analysis 237

Figure 4 Methylation of Calcitonin in colon tissue (left) and serum (right) of colon cancer patients and normal controls represented as percent methylated reference (PMR). conditions. However, the biopsies may miss the cancer gesterone receptor positive tumor samples from patients and, thus, fail to identify it. Therefore, a follow-on test to with recurrent breast cancer and recorded response to improve the sensitivity of the diagnosis is highly desira- Tamoxifen treatment and progression-free survival time ble. By combining the biopsy with GSTP1 methylation w26x. Five genes were identified, the methylation state of analysis, the sensitivity of the histologic biopsy review which correlated well with the Tamoxifen response. When was demonstrated to improve from 64% to 75% (both combined into a marker panel, the methylation of these at 100% specificity). The improved accuracy can help markers predicts the progression-free survival time much avoid the necessity for taking further needle biopsies, better than traditional predictive factors, as described by which represents a clear benefit to the patient. This dem- Ciocca and Elledge w25x. onstrates that DNA methylation analysis can also be The differentiation between responders and non- used to complement existing routine diagnostics. responders is only one of several pharmacodiagnostic applications which DNA methylation analysis is suited for. Patient and disease stratification In general, it can be used to analyze predictive markers linked to the sensitivity to specific therapies as well as One of the factors affecting the clinical outcome, e.g. in prognostic markers independent of treatment, such as cancer chemotherapy, is the response of the tumor to the aggressiveness of a disease. the drug. Efforts have been made to discover biomarkers enabling to access the molecular signature of responders and non-responders, e.g. in breast cancer. Such markers are of significant diagnostic value, as they are able to Outlook guide treatment decisions. A considerable portion of patients with primary breast cancer fail adjuvant endo- The clinical potential of DNA methylation analysis has crine therapy. 40–50% of patients with advanced disease been widely acknowledged. To date, methylation markers do not respond to endocrine therapy. Tamoxifen is one have been discovered for a variety of clinical questions of the most effective treatments for breast cancer. and the technology to analyze these markers in the rel- Tamoxifen, which is chemically similar to estrogen, evant clinical samples is in place. The role of DNA meth- blocks the reception of the estrogen message to its ylation in its interaction with other epigenetic phenomena receptor and, thereby, prevents cancer cells from grow- is increasingly well understood. Within the next years, the ing w24x. The choice of the right molecular markers to first methylation-based diagnostic tests will arrive on the predict response to Tamoxifen is difficult, since a whole market. Among them, there will be population-based ear- range of genetic and epigenetic mechanisms, such as ly screening tests, e.g. for colon, prostate and breast genetic instability, the emergence of receptor-negative cancer. Pharmacodiagnostic tests will provide physicians clones, blood supply and others can lead to resistance with essential information to help guide an appropriate to the drug w25x. In a recent study, the methylation of 117 therapy. The combination of diagnosis and therapy, candidate genes was studied in 200 estrogen and/or pro- based on this information and robust proprietary tech- 238 M. Schuster: DNA methylation analysis

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