Technical advances in global DNA methylation analysis in human cancers
© The Author(s). 2017
Received: 9 December 2016
Accepted: 10 February 2017
Published: 1 March 2017
Prototypical abnormalities of genome-wide DNA methylation constitute the most widely investigated epigenetic mechanism in human cancers. Errors in the cellular machinery to faithfully replicate the global 5-methylcytosine (5mC) patterns, commonly observed during tumorigenesis, give rise to misregulated biological pathways beneficial to the rapidly propagating tumor mass but deleterious to the healthy tissues of the affected individual. A growing body of evidence suggests that the global DNA methylation levels could serve as utilitarian biomarkers in certain cancer types. Important breakthroughs in the recent years have uncovered further oxidized derivatives of 5mC - 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), thereby expanding our understanding of the DNA methylation dynamics. While the biological roles of these epigenetic derivatives are being extensively characterized, this review presents a perspective on the opportunity of innovation in the global methylation analysis platforms. While multiple methods for global analysis of 5mC in clinical samples exist and have been reviewed elsewhere, two of the established methods - Liquid Chromatography coupled with mass spectrometry (LC-MS/MS) and Immunoquantification have successfully evolved to include the quantitation of 5hmC, 5fC and 5caC. Although the analytical performance of LC-MS/MS is superior, the simplicity afforded by the experimental procedure of immunoquantitation ensures it’s near ubiquity in clinical applications. Recent developments in spectroscopy, nanotechnology and sequencing also provide immense promise for future evaluations and are discussed briefly. Finally, we provide a perspective on the current scenario of global DNA methylation analysis tools and present suggestions to develop the next generation toolset.
Keywords5-methylcytosine (5mC) 5-hydroxymethylcytosine (5hmC) 5-formylcytosine (5fC) 5-carboxylcytosine (5caC) LC-MS/MS Immunoquantitation Next generation toolset
Background: The trail of DNA methylation derivatives
In 1866, Gregor Mendel published his seminal research detailing the laws of inheritance  and shortly afterwards in 1869 Friedrich Miescher discovered the enigmatic compound “nuclein” or DNA as we know it today . In the first half of the 20th century, Conrad Waddington designated the term “epigenetics” to describe “the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being”  and used the “epigenetic landscape” metaphor to describe events contributing to embryonic development . The “Sequence Hypothesis” proposed by Francis Crick in 1958  was ultimately established as the “Genetic Code” by research efforts of Marshall Nirenberg, Har Gobind Khorana and Robert Holley . While the genetic code lays out the procedure for translating hereditary information stored in DNA into functional attributes, the natural laws pertaining to “regulation of gene expression” or commonly referred to as the “epigenetic code” are still not understood. The explorative successes of post-1960 research have no doubt enhanced the current knowledge about the diversity of epigenetic mechanisms and its relevance in cancers [for a comprehensive understanding of the history of epigenetics refer to ], but as suggested by Bryan Turner much more needs to be done in terms of characterization of epigenetic marks and delineating their biological functions, before the epigenetic code can be deciphered .
Relevance of the expedition: Global “hypomethylation” in cancers is almost universal
The global loss of DNA methylation content in human tumors compared to normal tissues was reported in 1983 through independent research efforts of Feinberg et al. and Gamasosa et al. [18, 19]. This novel discovery was initially disregarded as “an unwelcome complication”  but research spanning the last three decades has confirmed that the trend of global hypomethylation in human cancers is almost universal [20–24], although each cancer type may have characteristic localized regions associated with hypermethylation or hypomethylation [25–27]. The association between global hypomethylation in cancers and the overarching loss of genomic integrity suggested by chromosomal abnormalities associated with mutations in DNMTs and misregulated methylation patterns over DNA damage repair genes/retroposon elements [9, 21, 28, 29] indicate that it is likely that these events contribute to maintenance of a catastrophic physiological state symptomatic of cancers.
Summary of representative clinical studies performed during the period (2011–2016) to estimate global levels of DNA methylation derivatives. Abbreviations: FFPE- Archived Formalin-fixed, Paraffin-embedded; IHC- Immunohistochemistry; Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS)
Method of Study
Clinical relevance of observation
Colorectal cancer (n = 30) Vs Control group (n = 30)
Loss of 5mC
Associated with advanced colorectal adenomatous polyps 
Clear cell renal cell carcinoma (n = 111) Vs matched adjacent tissue
Loss of 5hmC
No correlation with grade/prognosis 
Urothelial cell carcinoma (n = 55) Vs matched adjacent tissue
Loss of 5hmC
No correlation with grade/prognosis 
5mC & 5hmC
Clear cell renal cell carcinoma (n = 36) Vs paired normal
Loss of 5hmCNo change in 5mC
No correlation with grade/prognosis 
Tongue squamous cell carcinoma (TSCC) (n = 248) Vs Tumor adjacent normal (TAN) (n = 235)
Loss of 5hmC
Associated with the poor disease-specific survival in TSCC patients 
Renal Cell Carcinoma (n = 889) Vs age, gender, ethnicity matched control group (n = 889)
Loss of 5mC
Associated with risk of developing RCC 
Hepatocellular carcinoma (n = 208)
Loss of 5mC
Associated with poor disease free survival 
Leukocytes of Breast cancer patients (n = 384) Vs matched control (n = 384)
Loss of 5mC
Associated with occurrence of cancer regardless of hormone receptor status 
Colorectal cancer with liver metastases (n = 42) Vs matched primary (n = 24)
No correlation 
Laryngeal cancer (n = 72) Vs adjacent normal laryngeal tissue (n = 72)
Loss of 5mC in both groups
No correlation 
Prostate Cancer (n = 48) Vs adjacent benign (n = 48)
Loss of 5mC
No correlation with prognostic /pathologic grade 
Parathyroid carcinoma (n = 17) Vs Parathyroid adenoma (n = 43)
Loss of 5hmC
Diagnostic criterion for rare disease 
Bone marrow & Blood from AML (n = 206) Vs Healthy control
Wide range of 5hmC
High 5hmC levels associated with poor prognosis, low levels have no correlation 
Glioblastoma (n = 162) Vs healthy control (n = 66)
Loss of 5hmC
Marker for tumor infiltration zones 
Breast cancer (n = 59) Vs healthy control (n = 28)
Gain of 5caC
No correlation arrived at 
5mC, 5hmC, 5fC & 5caC
Colorectal carcinoma (n = 24) Vs matched tumor-adjacent normal
Loss of 5hmC, 5fC and 5caC. No change in 5mC
No correlation arrived at 
Undertaking the expedition: Tools for quantifying DNA methylation derivatives
In 2005, Song et al. reported a liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)  based method to quantitate 5mC and prescribed its application to archived tumor specimen as well as clinical samples derived from laser capture micro-dissection owing to a sensitive limit of detection (LOD) of 0.2 fmol and requirement of as little as 4 ng input DNA (Fig. 2a). Kok et al. further developed this method further and utilized the principle of LC-ESI-MS/MS to quantitate 5mC  and reported a LOD of 2 pg of cytosine and 5-methylcytosine. After the discovery of 5hmC in the human genome, it became imperative to include its quantitation to evaluate the global methylation landscape and Le et al. developed the liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneous quantitate the global levels of 5mC and 5hmC  with a LOD of 0.5 fmol per nucleoside base.
Recently developed methods based on variations in liquid chromatographic techniques have pushed the limit of epigenetic analysis and have subsequently been modified to include quantification of 5fC and 5caC in addition to 5mC and 5hmC. The discoverers of 5fC and 5caC, Ito et al. adapted the mass spectrometric parameters and reported the LOD to be 5 fmol and 10 fmol respectively . Thereafter to account for the low abundance of 5fC and 5caC, several modifications have been introduced to enhance the detection limits of these derivatives by LC-ESI MS/MS. In 2015, Tang et al. developed a labeling technique involving selective derivatization of cytosine moieties using 2-bromo-1-(4-dimethylamino-phenyl)-ethanone prior to LC-ESI-MS/MS for quantifying all the four known DNA methylated derivatives concurrently in archived Formalin-fixed Paraffin-embedded (FFPE) tumor specimen . The LOD of 5mC, 5hmC, 5fC and 5caC were described as 0.10, 0.06, 0.11, and 0.23 fmol respectively, representing a 35–123 fold enhancement in detection sensitivity compared to LC-ESI-MS/MS without chemical derivatization. In addition, Zhang et al. hydrolyzed genomic DNA by formic acid and analyzed 5caC by hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)  yielding an LOD of 0.1 ng/mL in the linear range of 40–4000 ng/ml. Yin et al. was able to demonstrate a 1.8 − 14.3 fold enhancement of the LC-ESI-MS/MS detection of 5fC along with 5mC and 5caC by the use of ammonium bicarbonate (NH4HCO3) as an additive to the mobile phase .
Despite the analytical superiority of LC-MS/MS, the key hindrances to its widespread use in quantifying methylated derivatives arise from the intricate procedures involved in analyzing and maintaining the instrument. Smaller clinics are particularly unwilling to adopt the LC-MS/MS technology owing to the exorbitant prices of the initial installation and the requirement of a highly skilled manpower to oversee daily operations. Compared to immunoassay based techniques which are commercially available in the form of kits with detailed working protocols, LC-MS/MS requires significant investment in terms of time and money in standardizing protocols. However, chromatography techniques are still the standard for pharmacokinetics and pharmacodynamics studies and will continue to dominate this field. Given the surge in epigenetics research we expect a significant effort in this field with emphasis on single cell analysis. Improvements in developing an automated workflow with technical support, will help lower service expenses, generate higher sample throughput and can have a considerable contribution in wider acceptance of LC-MS/MS.
Microtiter plate based immunoquantification known as enzyme-linked immunosorbent assay (ELISA) is a well-established method and can be effectively applied for the detection of epigenetic modifications of DNA immobilized on plastic, using an antibody highly specific to the target epigenetic marks (Fig. 2b). As early as 2000, Piyathilake et al. reported suitability of immunoquantification of 5mC over other first generation methylation quantification assays in rare clinical specimen . For 5mC, quantitative analysis by ELISA as well as the semi-quantitative immunohistochemical evaluation in clinical biopsies or cells collected by laser capture microdissection (LCM) offers the advantage of cost and speed. In 2012, Kremer et al. generated a rapid and sensitive ELISA based assay to quantitate 5mC (methDNA-ELISA) . This method requires as little as 10 ng of input genomic DNA, demonstrates linearity in the 1–10% genomic range and correlates well with MS approaches of 5mC quantification. Commercially available antibodies targeting 5hmC, 5fC and 5caC were practically nonexistent in the early years following their discovery, however post 2011 with the generation of highly specific antibodies, immunoassays were gradually adapted to include quantification of these novel epigenetic derivatives as well. Li et al. investigated for the first time the abundance of 5hmC in human tissues by ELISA . This method yields an LOD of 0.1 ng with a dynamic range 0.2–10 ng of 5hmC.
For simultaneous analysis of the four epigenetic marks, Chowdhury et al. designed a biotin–avidin mediated enhanced enzyme-based immunoassay (EEIA) and evaluated its performance in genomic DNA isolated from peripheral blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancers . Analytical sensitivity was significantly improved by increasing the number of labeling enzymes facilitating color detection on the antibodies, achieving a LOD of 1–2 pg and enabling the detection of the rare epigenetic marks. EEIA was subsequently utilized to evaluate the extent of alteration of the methylated DNA derivatives upon treatment by Decitabine- an FDA approved DNA demethylating drug in myeloid malignancies  as well as by the chemically induced hypoxia agent sodium dithionite , indicating the versatility of the assay in multiple contexts. Recently by utilizing the potential of locus specific methylation status to confer conformational differences, Kurita et al. introduced a novel immunochemical approach of performing methylation analysis at single CpG loci on a conventional microtiter plate format . Microtiter plate assay is universal and commercialized by biotech companies such as Epigentek Group, Sigma-Aldrich and Zymo Research. However, the analytical sensitivity of the rarer epigenetic derivatives particularly 5fC and 5caC is variable and often these derivatives remain undetectable. Sample processing and the unknown biological context of these derivatives may in some ways contribute to the unpredictability in detection of these rare marks. Key opportunities to advance this technology is in requiring less input DNA to perform the analysis as well as incorporation of a suitable signal enhancement strategy using well-defined conjugates including nanoparticles, enzymes and fluorophores. Given the familiarity of immunoquantification tools, this approach will continue to be extensively used in methylation analysis primarily due to the relative ease of adaptability in a clinical setting.
There have been many interesting reports on fluorescence-based epigenetic analysis owing to its simplicity for signal generation and detection. Wang et al. demonstrated a particle counting assay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts that were captured by immuno-magnetic particles . By amplifying fluorescence signal with OliGreen™ dyes, the captured BPDE-DNA adducts could be quantified by particle counting, which was proportional to the modification level in genomic DNA. The detection of limit was about 180 fM. In addition, Feng et al. developed a fluorescence resonance energy transfer (FRET) assay using an optically amplifying cationic conjugated polymer (CCP, poly((1,4-phenylene)-2,7-[9,9-bis(6′-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide)) . The occurrence of FRET between CCP and fluorescein (Fig. 2c) incorporated into DNA was used for read out, however this assay took about 20 h to attain the methylated level of cancer cells. Zhang et al. utilized an identical method for diagnostic and screening of cancer . Single molecule techniques to monitor the dynamics of epigenetic proteins exist [57, 61] but these are not applicable for routine analysis. Precedence for quantification of epigenetic marks in nucleosomes including resolving the stoichiometry of the epimarks using single cell-based FRET approaches also exist  and these tools remain to be optimized for DNA methylation analysis. With advances in microscopy, especially in sensitivity (single molecule techniques) and resolution (super-resolution techniques), basic research will continue to enhance our understanding of the dynamics of epigenetic programming.
As one of the highly sensitive spectroscopic techniques, Surface plasmon resonance (SPR) known for its appeal in monitoring biomolecular interactions have also been applied in epigenetics evaluation (Fig. 2d). Nguyen et al. introduced a strategy for ultrasensitive detection of methylation of ctDNA of PIK3CA gene based on localized SPR (LSPR) associated with plasmon coupling mode of gold nanoparticles to observe a shift in the LSPR peak upon the immunogold colloids binding to two methylcytosines, to yield an extremely low LOD of ~50 fM. Kurita et al. reported a sequence-specific immunoassay chip for DNA methylation by microfluidic surface plasmon resonance (SPR) detection . By utilizing an affinity measurement involving the target, (methyl-) cytosine, in a single-base bulge region and an anti-methylcytosine antibody in combination with a biotinylated bulge-inducing DNA probe, this system could obtain the methylation status in 6 attomoles (48 femtograms) of synthesized oligo DNA in 45 mins, which is the fastest DNA methylation assessment hitherto reported. Darkfield microscopy based on SPR have been implemented by Wang et al. to quantify global methylation levels at the single cell level , showing promise as a routine screening tool for in situ analysis in the context of tissues.
Variations of electrochemical tools based on redox reactions have been introduced for detection of DNA methylation. Kurita et al. introduced methylated cytosine in DNA via ELISA with ECL detection in real genomic sample  (Fig. 2e). Here, an acetylcholinesterase was employed as enzyme tracer labeled with anti-methyl cytosine, which converted acetylthiocholine to thiocholine, enabling accumulation on gold electrode surface and quantitatively measurement of 5mC in the range from 1 to 100 pmol. By glycosylation modification of 5hmC with glucosyltransferase and sodium periodate, Chen et al. detected 5hmC at sub-nanogram level, which was 20 times more sensitive than the commercial kit based on optical measurement . Carbon-based nanomaterials such as carbon nanotube and graphene were recently employed as alternative electrodes to the conventional metal electrode due to its high electrical conductivity. Wang et al. reported a polypyrrole (PPyox)-directed multiwall carbon nanotubes (MWNTs) film modified glassy carbon electrode (GCE) which was used to electrically oxidize DNA bases for evaluation of DNA methylation level . Due to extraordinary catalytic property of PPyox/MWNTs/GCE, the peak potential of 5mC was distinctive compared with other bases, especially the unmethylated cytosine, upon applying 180 mV, enabling rapid detection of the methylation status in real samples within 45 min. The major advantage of electrochemical method is limit of detection and miniaturization. Additionally, it can be anticipated that micro-electro-mechanical system (MEMS) and nanotechnology will be combined for miniaturization in the future. However, the lower sample volume may cause low signal-to-noise ratio, thus more elaborate manufacturing process is required.
Microfluidic platform technology has several advantages over conventional analytical methodologies, enabling fast response, cost effectiveness and low consumption of reagents. Recently this method has been applied in the field of epigenetics to efficiently enhance performance of DNA methylation analysis. Cipriany et al. used fluorescently labeled Methylated DNA Binding Domain (MBD) proteins as probes to perform Single-Chromatin analysis at the nanosacle (SCAN) in DNA restricted to microfabricated nanofluidic channels (Fig. 2f) enabling rapid and real-time interrogation of individual molecules of methylated DNA based on their fluorescent signatures . Ronen et al. presented a universal, high-throughput, microfluidic-based fluorometric method for studying DNA methylation , employing bacterial HPAII DNA methyltransferase of which enzymatic activity was analyzed by measuring Michaelis-Menten constant. The values were determined to be 5.8 nM and 9.8 nM respectively. These pioneering efforts paved the road to the realization of epigenetic analysis in microfluidic devices, with a possibility of ultimately utilizing these devices in point-of-care testing. However, despite its advantages over conventional methods, limited work exists in microfluidic-based epigenetic analysis. A possible reason could be the complexity of sample preparation, reliability and robustness of the approach.
Nanopore technology offers a promising alternative to conventional DNA sequencing by measuring distinctive electric currents obtained from different bases and has been recently applied for epigenetic studies. Zeng et al. reported a α-hemolysin-based nanopore sensing method (Fig. 2g) for 5mC and 5hmC detection in DNA at the single-molecule level . Here, 5hmC is first selectively modified with iron-linked crosslinker via Click chemistry. Subsequently the passage of the modified bases through nanopores causes unbinding of the host-guest complex generating characteristic current signatures and enables obtaining quantitative information on the 5mC and 5hmC. Recent studies have focused on evolving an electronic signature of methylated DNA bases  as well as development of novel nanaomaterials for fabricating nanopores. The electronic signature based monitoring of modified DNA bases through nanopore has excellent appeal in high throughput, especially considering the state-of-art standardization of manufacturing process in materials research.
Nanoparticle based tools
Nanoparticles that have been employed as tracers in many biosensor applications have also been employed in exploratory epigenetic research owing to its extraordinary physical properties such as photothermal effect, localized surface plasmon resonance which are based on electromagnetic field passing around the nanoparticle surface. The essence of this approach is the induction of particle aggregation to observe a shift in peak for detection by fluorescence or colorimetry. Ge et al. demonstrated a simple colorimetric method to detect DNA methylation . Here, methylated CpG region was captured and enriched by immunomagnetic separation followed by release via heat denaturation. By controlling salt-induced aggregation process associated with unmodified gold nanoparticle, a LOD of 80 fmol was achieved. This method is semi-quantitative by common UV/Vis spectrophotometer, enabling simple and rapid detection of DNA methylation. Interestingly, nanoparticles can be utilized for enhancing efficiency of isolation of genomic DNA and can be subsequently utilized for methylation analysis. Zhou et al. developed a novel one-point extraction technique from whole blood employing bi-functional carboxyl-functionalized magnetic nanoparticle used as solid-phase adsorbent . Here, the extracted chromatin from leukocytes via magnetic separation was concentrated and coated on a microtiter well and analyzed  for the detection of the four different cytosine derivatives. Nanoparticles depending upon the material can be used as reporters in a sensor device. Since the size can be tuned in the range from 10 to 200 nm with slight modification of existing protocols, there are many ways to optimize analytical conditions for epigenetic analysis and utilization of nanoparticles in other detection modalities, such as microfluidics, plasmonics and electrochemical sensing, and in spectroscopy. Fig. 3a demonstrates the basic concept of nanoparticle-based aggregation as a signal for SERS and LSPR sensing.
Surface enhanced Raman scattering (SERS) based tools
Wang et al. developed a novel concept for enzymatic control of plasmonic coupling for DNA demethylation . Here, gold nanoparticle with a Raman reporter and hemimethylated DNA were used as probes. Destabilized nanoparticles were aggregated among others, which generated strongly distinctive SERS signals in response to DNA methylation. Since this method was performed by a homogenous single step analysis, rapid, convenient and a miniaturized analytical method for epigenetic analysis could be developed. (Fig. 3b) Furthermore silver nanoparticles were also used as SERS-based enhancement substrate combined with hybridization chain reaction for the determination of DNA methyltransferase . Morla-Folch et al. demonstrated the feasibility of direct SERS in combination with chemometrics and microfluidics for the relative quantification of the four DNA methylation derivatives in single- and double-stranded DNA . More recently, Ouyang et al. have shown that detection of 5caC and 5hmC along with 5mC is possible with SERS using a novel graphene wrapped plasmonic material . In the future, enhanced approaches based on nanoparticles or enzymes or development of hand-held units will be more common place. Given the recent work and the advent of new materials and standardization of manufacturing processes, one can expect SERS to become a viable option for routine monitoring of epigenetic events.
Conclusion and the future roadmap
It is also conceivable that with the emergence of Next Generation Sequencing (NGS) technologies, quantification of global methylation derivatives along with the precise identification of localized sites undergoing these alterations will become prevalent. While this may in the foreseeable future help clinicians make informed choices pertaining to patient profiling and therapeutic management, standards will have to be developed to decorously interpret the disease risk imparted by global changes of the methylome. We realize that the infrastructural wealth available to scientists in biomedical settings may not be practical in a clinic and in this regards, and to address this challenge our lab and others have used lateral flow techniques that can potentially be used for onsite sensing [85, 86] in conjunction with chromatin extraction methods  to addressing this lacunae. Sample preparation will continue to challenge the point of care sensors development. However, we are optimistic that advances in miniaturization, development of novel materials, production of capture biomolecules (antibodies, aptamers etc.) will infuse sufficient enthusiasm to further the field of developing analytics for epigenetics. Finally, further explorations of the molecular dynamism of 5hmC, 5fC and 5caC will bring clarity to their biological significance in cancers and identify other areas for the development of tools for diagnostic determination of the methylated DNA derivatives. We expect loci-specific evaluation and quantification of epigenetic targets utilizing modern technologies to become important metrics with more mechanistic studies. In this regard, development of algorithms with heuristics to expound on the profiles of methylome for prognostic determination could become prominent. In summary, it is exciting to note the milestones covered in this trail of DNA methylated derivatives and assess from these studies the impending way ahead for developing tools that hold the key to understanding the “epigenetic code” and its deregulation in diseases such as cancer.
Dr. Yi Cui for his help in preparing Fig. 3b.
W.M. Keck Foundation, the Indiana-CTSI and Purdue Center for Cancer Research.
Availability of data and materials
B.C. (biological), I.H. (technical) and J.I. drafted and approved the final manuscript.
The authors declare that they have no competing interests.
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