DNA Methylation Biomarkers for Cancer – a Measurement Challenge

Although studied for decades, the study of methylated cytosine DNA residues is not a trivial task for clinical in vitro diagnostics

The challenge of methylation analysis in clinical diagnostics

The challenge of methylation analysis in clinical diagnostics

In the world of diagnostics, the measurement of biological analytes for clinical use trace its origins back to 1919 when Otto Folin and Hsien Wu devised a test to measure serum glucose concentration using alkaline copper reduction to produce a blue colorimetric indicator. (Reference 1) Today there are literally thousands of analytes tested for a myriad of health conditions, and if you want to count rare disease genetics (and rare causal variant diagnostics for newborns for example) that number may well be in the hundreds of thousands.

The quantitative measurement of methylation of CpG residues is not a trivial task for the clinical application of methylation for in-vitro diagnostics. Nonetheless interest in methylation grows, particularly for cancer diagnostics and early detection; here we will look at what challenges the clinical molecular laboratory needs, the limitations of methylation-detection methods, and assess the current state of methylation analysis in clinical molecular diagnostics.

Clinical requirements for molecular diagnostics

In the modern clinical testing laboratory for diagnostics, there are a wide variety of completely automated instruments for measuring a plethora of analytes. One major central laboratory offers over 3,000 different analytes, excluding the dozens that are offered within the hospital environment (called point-of-care or point-of-need tests).

For molecular diagnostics, there is a higher level of training required as these molecular tests are not nearly as automated. Techniques such as PCR, real-time PCR, and Sanger sequencing are expanding in its use and levels of automation, including sample preparation (purification of DNA or RNA from the sample). Offerings such as from the molecular diagnostics companies Cepheid or Hologic have improved the level of ease-of-use for PCR and real-time PCR applications, however other molecular methods including next-generation sequencing require a remarkably high amount of manual manipulations.

The handling of the information coming from these instruments is also important – integration of LIMs (laboratory information management systems), data security, and how the data flows back to the clinician ordering the test.

With the increasing use of NGS in the molecular laboratory, a set of challenges pose themselves across these areas – from the training of the laboratory technician to the amount of automation to adopt (including sample preparation and sequencing library preparation) to the handling of the immense amount of data produced by a multi-gene test.

Included with these challenges come the laboratory’s need for analytical validation, as many of the tests offered are labeled For Research Use Only (i.e. have not been cleared by the FDA) and need to be offered as a Laboratory-Developed Test under CLIA/CAP guidelines. For more information, Center for Medicaid Services (CMS) has put together this CLIA Laboratory Developed Test FAQ (PDF) to show how the LDT and FDA clearance are complementary processes. >

In the course of an analytical validation of an LDT, it is important to note the clinical validation is out of scope of the LDT in question.

From the Genetics Home Reference: “How can consumers be sure a genetic test is valid and useful”:

  • Analytical validity refers to how well the test predicts the presence or absence of a particular gene or genetic change. In other words, can the test accurately detect whether a specific genetic variant is present or absent?
  • Clinical validity refers to how well the genetic variant being analyzed is related to the presence, absence, or risk of a specific disease.
  • Clinical utility refers to whether the test can provide information about diagnosis, treatment, management, or prevention of a disease that will be helpful to a consumer.

Therefore laying aside the clinical validity of methylation as a biomarker, for its analytical validity methylation analysis has been studied for decades yet faces challenges with its clinical implementation, limiting its uptake.

Indirect methods of measuring methylation using bisulfite

In ordinary PCR, the DNA polymerase does not distinguish between methylated and unmethylated cytosines, thus the original DNA template must be chemically modified to measure the level of methylation. The first paper referring to the bisulfite method dates back to 1992. (Reference 2)

While there are direct methods of measuring methylation (e.g. using a methylation-sensitive restriction enzyme and comparing it to a methylation-insensitive is one of several approaches), the bisulfite method is considered to be the current ‘gold standard’. The use of PCR with bisulfite-treated DNA as a template ‘are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci’. (Reference 3) The downside of the bisulfite method, however, is the amount of damage that occurs with bisulfite treatment through the use of acidic conditions as part of the reaction (Reference 4).

Bisulfite Conversion Figure Hayatsu Mutat Res. 2008 PMID: 18485805

Bisulfite Conversion Figure Hayatsu Mutat Res. 2008 PMID: 18485805

In brief, the original CpG sites across the genome that are unmethylated cytosine residues are chemically converted to uracil while the methylated cytosines are protected. During sequencing (or depending on the method during the PCR before the sequencing itself), the converted uracil base is read as a thymine base, and in the sequence analysis appears to be a C -> T conversion. This bisulfite conversion reaction is a balance between the complete conversion of all the unmethylated cytosine bases to uracil, and the breakage and removal of the sugar residues (the process of depurination creating abasic sites) in the process. (Reference 4)

Current clinical diagnostics for cancer and their related genes

There are three types of cancer with CpG methylation diagnostic tests associated with them: colorectal, lung, and prostate cancer; also there is a prognostic test for glioblastoma. These tests have been all developed with the use of bisulfite treatment, and mainly use real-time PCR as the downstream analysis, with one using pyrosequencing. (Reference 5)

Commercially available DNA methylation tests for cancer

Commercially available DNA methylation tests for cancer

The difficulty to translate research findings into a clinical test has been complicated by the aforementioned realities of the clinical molecular laboratory – the more equipment, the additional steps and manipulation create barriers, not to mention concerns for clinical validity and clinical utility, and laboratory test reimbursement. Thus the tests listed above are primarily offered as Research Use Only test kits, with the only two that are FDA-approved are the VIM/SEPT9-based Cologuard test from Exact Sciences for colorectal cancer screening (offered as a service) and the SEPT9-based Epi proColon test from Epigenomics AG (offered as a service but recently announced a kit recently).

Singlera’s approach to methylation analysis

Can the bisulfite-treatment method be improved? NEB has been working on an enzymatic approach to conversion, which is still in development and yet to be commercially available. Can NGS be made easier to use and implement? New platforms are on the horizon, including NanoString and (even) GenapSys.

In the meantime, we at Singlera will be using bisulfite treatment with cell-free DNA, coupled with next-generation sequencing as a readout platform. The barriers to clinical adoption remain, with the need for the bisulfite manipulations along with the need for NGS, which poses its own challenges within the clinical molecular diagnostics laboratory. However due to the richness of the data produced, and the high levels of sensitivity and specificity produced so far (refer to our assay page for details) the effort to adopt this technology is well worthwhile.


  1. Folin O and Wu H. A system of blood analysis. J Biol Chem 1919 http://www.jbc.org/content/38/1/81
  2. Frommer M and Paul CL et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 1992 PMID: 1542678 https://www.ncbi.nlm.nih.gov/pubmed/1542678
  3. Kristensen LS, Hansen LL. PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem. 2009 PMID: 19520761 https://www.ncbi.nlm.nih.gov/pubmed/19520761
  4. Hayatsu H. The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research. Mutat Res. 2008 PMID: 18485805 https://www.ncbi.nlm.nih.gov/pubmed/18485805
  5. Mikeska T, Craig JM. DNA methylation biomarkers: cancer and beyond. Genes (Basel) 2014 PMID:25229548 https://www.ncbi.nlm.nih.gov/pubmed/25229548