PERSpECTIVES

(Table 1; Supplementary Table 2). Of these

OPINION
14 biomarker assays, only nine (GSTP1,
APC, RASSF1, NDRG4, BMP3, two SEPT9
Analysis of DNA methylation in biomarkers, SHOX2, and MGMT) have
been included in one or more clinical
cancer: location revisited guidelines2–7. Moreover, only two tests have
received FDA approval: Cologuard (NDRG4
and BMP3) for the analysis of stool DNA
Alexander Koch   , Sophie C. Joosten, Zheng Feng, Tim C. de Ruijter, samples collected as part of colorectal cancer
Muriel X. Draht, Veerle Melotte, Kim M. Smits, Jurgen Veeck, screening protocols and Epi proColon
James G. Herman, Leander Van Neste, Wim Van Criekinge, Tim De Meyer and (SEPT9) for analysis of blood samples taken
for the same purpose. This rate of translation
Manon van Engeland
of ~0.8% (14 commercially available DNA
Abstract | Changes in DNA methylation in cancer have been heralded as promising methylation-​based assays versus an estimated
targets for the development of powerful diagnostic, prognostic, and predictive 1,800 reported in various publications) is in
line with the numbers presented by George
biomarkers. Despite the existence of more than 14,000 scientific publications Poste8 in 2011 (100 biomarkers used in the
describing DNA methylation-​based biomarkers and their clinical associations in clinic versus 150,000 biomarker-​related
cancer, only 14 of these biomarkers have been translated into a commercially publications) and Scott E. Kern9 in 2012
available clinical test. Methodological and experimental obstacles are both major (<1% of cancer biomarkers reported in
causes of this disparity , but the genomic location of a DNA methylation-​based publications reach the clinic) (Fig. 1).
The above-​mentioned translational
biomarker is an intrinsic and essential property that also has an important and
success rate and the current public
often overlooked role. Here, we examine the importance of the location of DNA discussion on the reliability and efficiency
methylation for the development of cancer biomarkers, and take a detailed look of scientific research10 warrant a reflection
at the genomic location and other relevant characteristics of the various on why so few DNA methylation-​based
biomarkers with commercially available tests. We also emphasize the value of biomarkers are transitioned from initial
publicly available databases for the development of DNA methylation-​based discovery and publication to a clinical
test. The answer to this question is
biomarkers and the importance of accurate reporting of the full methodological complex, multifaceted, and mainly
details of research findings. relates to small effect sizes and a lack of
substantial clinical value, as well as various
In 2003, Peter Laird1 expressed his features of cancer (the query was executed methodological caveats (such as biased
belief that the then recent advances in in January 2018, Supplementary Table 1). patient selection, improper study design
our understanding of the role of DNA This search returned 14,743 research articles, and data analysis, lack of validation, and/
methylation in human cancer could one representing an estimated 1,800 markers, on or inappropriate reporting) that prevent
day result in a multitude of powerful the basis of manual curation of representative the thorough evaluation of the clinical
DNA methylation-​based biomarkers, subsets of these publications. However, only value of a biomarker. Furthermore, the
particularly for use as cancer diagnostics. 14 DNA methylation-​based biomarker assays financial investment needed to develop
This belief was based on a number are currently commercially available and are an assay quantifying a specific biomarker
of characteristics of changes in DNA designed to measure the methylation of only or biomarkers, including trials providing
methylation that make them promising 13 genes in total: glutathione S-​transferase P validation and exploring clinical utility, is a
biomarkers: early and frequent occurrence (GSTP1); adenomatous polyposis coli protein major obstacle. These issues are not unique
in cancer, easy detection by well-​ (APC); ras association domain-​containing to DNA methylation-​based biomarkers.
established techniques, stability of DNA protein 1 (RASSF1); N-​myc downregulated Researchers in the field of biomarker
methylation in fixed samples over time, gene 4 (NDRG4); bone morphogenetic research in general face similar problems,
presence in various bodily fluids, and protein 3 (BMP3); septin-9 (SEPT9); short although only now are such problems
cell-​type specificity. stature homeobox protein 2 (SHOX2); twist-​ beginning to be addressed8,9,11,12.
Interest in changes in DNA methylation related protein 1 (TWIST1); homeobox Apart from the established issues
as cancer biomarkers has resulted in a large protein OTX1 (OTX1); one cut domain surrounding clinical translation, we
number of scientific publications. To more family member 2 (ONECUT2); methylated-​ believe another often overlooked barrier
accurately define this number, we performed DNA-protein-​cysteine methyltransferase exists between the discovery and clinical
a search of the PubMed database for (MGMT); branched-​chain-amino-​acid implementation of DNA methylation-​based
publications that describe DNA methylation-​ aminotransferase, cytosolic (BCAT1); and biomarkers in patients with cancer. In 2011,
based biomarkers, in relation to clinical DNA-​binding protein Ikaros (IKZF1) we addressed the importance of the genomic

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PerspeCTIves

Table 1 | Commercially available assays designed to measure Dna methylation-​based cancer biomarkers
genes indication Hypermethylated Correlation with Promoter Cpg Clinical FDa Refs
in tumour? gene expression? region? island? guideline? approved?
ConfirmMDx
GSTP1 Diagnosis and prognosis of Yes Yes Yes Yes Yes2 No 57

prostate cancer
APC Yes No No No
RASSF1 Yes No Yes Yes
Cologuard
NDRG4 Early detection of CRC Yes Yes Yes Yes Yes3–5 Yes 77,78

BMP3 Yes Yes Yes Yes

Epi proColon/ColoVantage (Licensed to Quest Diagnostics and ARUP Laboratories) a

SEPT9a Early detection of CRC Yes No No Yes Yes4,7 Yes 79

Epi proColon a

SEPT9a Early detection of CRC Yes No No Yes Yes4,7 Yes 80

Epi proLung
SHOX2 Early detection of lung cancer Yes Yesb No Yes No No 81

AssureMDx
TWIST1 Risk of bladder cancer in Yes Yes Yes Yes No No 82

patients with haematuria

OTX1 Yes Yes b
No Yes
ONECUT2 Yes Yes b
No Yes
PredictMDx
MGMT Prediction of response to NAc Yes Yes Yes Yes6 No 20,83

temozolomide in glioblastoma
Colvera
BCAT1 Early detection of CRC Yes Yes Yes Yes No No 84

recurrence
IKZF1 Yes Yes Yes Yes
The Cancer Genome Atlas (TCGA) data for all biomarkers with commercially available assays were analysed for differential methylation between tumour and
nonmalignant samples and to determine whether or not methylation status at the location of the area covered by the assay is correlated with expression. We also
investigated whether or not the biomarkers are located in the promoter region of their corresponding genes and whether or not they are located in a CpG island.
Data on the clinical performance of these biomarkers, as reported in the literature, are listed in Supplementary Table 2. CRC, colorectal cancer ; NA , not available.
a
Two different SEPT9 biomarkers are available, which both target the same genomic region but at slightly different locations. In some publications, the SEPT9
biomarker is variably referred to as the ColoVantage test by Quest Diagnostics, as a test provided by ARUP Laboratories, or as the Epi proColon test provided by
Epigenomics. Both biomarkers have been patented by Epigenomics83,85, which has licensed one of them to Quest Diagnostics and ARUP Laboratories. bA positive,
instead of a negative, correlation exists between DNA methylation and gene expression of SHOX2, OTX1, and ONECUT2. cMGMT is a predictive marker, rendering
the comparison between tumour and nonmalignant samples less relevant. Furthermore, no nonmalignant samples of brain tissue are available in TCGA for
comparisons with glioblastoma specimens.

location of DNA methylation in relation to
gene expression and/or clinicopathological
characteristics of cancers and emphasized
how crucial this aspect is for biomarker
development13. We argued that both a better
understanding and more detailed analysis
of the clinical relevance of the genomic
location of DNA methylation is required to
increase the number of such biomarkers that
can be successfully implemented in patient
care. In this Perspective, we hypothesize
that epigenomic data that are currently
publicly available offer an opportunity to
better select the optimal location of DNA
methylation-​based biomarkers. We closely
examine the genomic location and other
relevant characteristics of DNA methylation-​
based biomarkers with potential for clinical
use in patients with cancer and for which an
assay is currently commercially available,
and conclude with recommendations for the
development of DNA methylation-​based
biomarkers that might improve the currently
limited translation of basic research into
clinical advances.
The importance of location
Traditionally, DNA methylation research
in general and DNA methylation-​based
biomarker studies have predominantly been
focused on the effects of hypermethylation
of promoter CpG islands in tumour-​
suppressor genes14. However, even within a
single promoter region, not all CpG islands
are functionally equivalent. Transcriptional
silencing, for example, is often controlled
by the methylation of one or more small
parts of a promoter, rather than by the entire
promoter region15–19. Thus, identifying
the precise location of clinically relevant
CpG islands is an important step in the
development of a DNA methylation-​based
biomarker, as illustrated in the following
examples.
Diagnostic methylation markers.
Methylation of GSTP1 has been reported
as a promising diagnostic marker for
hepatocellular carcinoma (HCC) in several
studies, but with widely varying levels of
specificity. This variability is hypothesized
to result from analysis of different specific
parts of the same promoter20. One group of
investigators conducted a detailed analysis
of methylation of the GSTP1 promoter
using bisulfite sequencing and found
that methylation of the 5ʹ region of the
promoter was significantly more specific
in distinguishing HCC from nonmalignant
liver diseases or liver tissue with no

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 PerspeCTIves

Number of biomarkers survival in four independent patient series

used in the clinic (all P ≤ 0.03)25.

14 Predictive methylation markers. In the

(0.8%)
initial studies that described the predictive
100 power of MGMT methylation in patients
(0.07%) <1% with glioblastoma, the methylation status
of MGMT was assessed using methylation-​
specific PCR (MSP), using primers covering
only a few CpG dinucleotides located
in the first exon and promoter regions of
the gene26–30. Direct correlations between
14,743
Published papers methylation and MGMT expression and/or
describing DNA- activity were not investigated. Following this
150,000 methylation based 100% finding, numerous investigators25–27 analysed
biomarker papers biomarkers cancer biomarkers
(Poste, 2011) (Kern, 2012) the promoter region of the MGMT gene in
~1,800 more detail, using quantitative techniques
biomarkers such as pyrosequencing31–33. The goal of
(Koch et al., 2018)
these studies was to gain greater insight into
specific methylation patterns, to identify the
Fig. 1 | Success rates for the clinical implementation of cancer biomarkers. The numbers of puta- CpG dinucleotides that are most important
tive biomarkers and related publications have been compared with the number of biomarkers used in in transcriptional regulation, and to identify
the clinic, with similar results obtained from different sources. The success rate of DNA methylation-​
the region or regions in which analysis of the
based cancer biomarkers (0.8%) was calculated by comparing the number of commercially available
biomarkers with the estimated total number of published biomarkers. A comprehensive search of the changes in methylation provides the highest
PubMed database for publications related to DNA methylation-​based cancer biomarkers returned clinical value. Conflicting results regarding
14,743 research papers. By counting the number of unique gene symbols in the corresponding the specific methylated region or regions,
abstracts (5,090), manually curating three random subsets of these abstracts (100 abstracts each) and or even individual CpG dinucleotides,
calculating the average proportion of cancer DNA methylation-​based biomarkers in these three sub- that correlate best with MGMT expression
sets (36.5%), we arrived at our estimate of ~1,800 unique biomarkers. The estimates of the translational have been published31–33. The region
success rate by Poste2 and Kern3 were derived based on the number of biomarkers used in the clinic encompassing CpGs 75–78, which is covered
versus the number of biomarker papers and the number of cancer biomarkers used in the clinic versus by the commercially available PredictMDx
the number of published biomarkers, respectively ; the exact calculation was not described in detail test, is currently the best studied and
by either author.
methylation of CpG dinucleotides in
this region has been repeatedly shown
established pathological alterations than investigators described an association to be associated with favourable patient
methylation of the 3ʹ end of the promoter between methylation of a small region prognosis33. This MGMT test has received a
(97.1% versus 60%; P < 0.001)20. The same in the promoter of the NMDAR2B gene, Tier 1 reimbursement code by the American
group obtained similar data on RASSF1 located just outside a CpG island, and Medical Association and was included in
methylation as an early detection marker for inferior overall and disease-​specific survival the 2013 National Comprehensive Cancer
HCC (72.9% specificity for promoter region outcomes in patients with oesophageal Network guidelines6, indicating that the test
1 compared with 27.1% for promoter region squamous cell carcinoma (HR 3.13, 95% should be widely used; however, the debate
2 and 38.6% for exon 1 at a fixed sensitivity CI 1.05–9.72; P ≤ 0.006), while DNA on the exact region of the promoter at which
of 90%; P < 0.0001)21. methylation at two other locations, located methylation provides the highest level of
further downstream of the TSS and within clinical value continues.
Prognostic methylation markers. In CpG islands, was not correlated with
previous research, we analysed three patient outcomes24. Genomic context
different genomic regions within CpG Interestingly, the methylation status of Methylation of specific CpG dinucleotides
islands of the GREM1 promoter and found single CpG dinucleotides, and not only within a single genomic region, such as a
that only methylation of CpGs in one of of wider regions that encompass multiple gene promoter, can regulate transcription
these regions was associated with inferior CpG dinucleotides, has been shown to as well as determining the clinical value of a
prognosis in patients with clear cell renal influence the regulation of gene expression biomarker. Therefore, identifying the most
cell carcinoma (RCC; hazard ratio (HR) and, therefore, the prognostic value of relevant genomic location of a potential
2.32, 95% CI 1.52–3.53; P = 0.001)22. Similar a DNA methylation-​based biomarker25. DNA methylation-​based biomarker is
results were observed in patients with A high-​resolution quantitative analysis important. Apart from location, the genomic
gastric cancer, in which hypermethylation of methylation of the entire ZAP70 gene context of DNA methylation including, for
of CpGs in a specific promoter region in samples from patients with chronic example, the presence of different functional
located close to the transcription start lymphocytic leukaemia (CLL) demonstrated regions, such as exons or enhancers, must
site (TSS), but not of CpGs in a different that methylation of a single CpG also be taken into account. The presence
location, in the MAL promoter was dinucleotide (located +223 from the TSS) is of these different regions is associated
associated with superior disease-​free particularly important for the transcriptional with characteristic methylation profiles.
survival (P = 0.03) and downregulation repression of ZAP70 and that methylation of Promoter CpG islands, for example, are
of gene expression (P = 0.01)23. Other this CpG is correlated with superior patient typically not methylated, whereas most gene

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PerspeCTIves
bodies, particularly exons, are extensively
methylated34. Research published in 2012
shows that alterations in DNA methylation
in both intergenic regions and intragenic
regions are modulated in development and
disease and that these alterations are actively
involved in the regulation of transcription35.
Methylation at non-​promoter sites has
a role in the regulation of several vital
processes, including splicing, the expression
of alternative transcripts from alternative
promoters, and enhancer activation,
although such DNA modifications remain
a largely unexplored area for biomarker
development. However, potential clinical
utility has already been demonstrated for
methylation-​based biomarkers in certain
non-​promoter locations. For example,
patients with gastric cancer who are positive
for long-​range epigenetic silencing (LRES),
which encompasses multiple neighbouring
genes, of the chromosomal region 15q25
have been shown to have a lower risk
of disease recurrence (HR 0.6; 95% CI
0.38–0.94; P = 0.027)36. Aberrant enhancer
hypermethylation has also been shown to
be prognostic for survival in patients with
RCC (log-​rank P < 0.05)37. With advances
in our understanding of the biology and
consequences of these types of DNA
methylation, we will undoubtedly see an
increase in the amount of published data
available on the role of methylation-​based
biomarkers located outside of promoter
regions. Similar to methylation-​based
biomarkers located in promoter regions, the
development of non-​promoter methylation-​
based biomarkers will also face the challenge
of determining the precise genomic region
associated with changes in gene expression
and/or clinical outcomes.
Biomarker development
For methylation of a specific genomic
location to be a good biomarker, a clinically
significant difference in methylation
between the groups of interest has to
be detected: for example, in tumour
versus nontumour tissue for a diagnostic
biomarker; between tumour samples from
patients with high-​risk disease versus low-​
risk disease for a prognostic marker; or
between responders versus nonresponders
for a predictive marker. The associated
gene should — ideally — be differentially
expressed between both groups. A classic
example of a candidate diagnostic biomarker
would be a hypermethylated CpG-​rich
region of the promoter of a tumour
suppressor gene that is downregulated in
tumour cells. We believe that these features
(a promoter containing hypermethylated
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
CpG islands in tumour cells accompanied
by downregulation of the associated gene)
reflect the current best practice for the
development of DNA methylation-​based
biomarkers. A link between methylation and
gene expression provides a clear biological
rationale, although it is important to note
that a DNA methylation-​based biomarker
does not necessarily have to be linked with
a measurable change in gene expression.
Of the commercially available biomarkers
for example, four (APC, RASSF1, and
both SEPT9 biomarkers) do not show a
correlation between DNA methylation and
expression of the associated gene (Table 1).
Cancer-​associated hypermethylation
can occur on silent genes marked by the
presence of bivalent chromatin, thus
preventing their activation rather than
actively suppressing their expression38,39.
This blocking of activation still implies a
link with expression even if no statistical
association is observed between the DNA
methylation status of a gene and its basal
level of expression. Unravelling such an
association would require additional
analyses such as re-​expression experiments
involving a demethylating agent or the
analysis of different tissues.
Other important considerations in
the development of a methylation-​based
biomarker include cell-​type specificity and
the optimal number of CpG dinucleotides to
cover. For example, methylation of potential
cancer biomarker genes has been observed
in peripheral blood samples containing
no malignant cells40, thus emphasizing the
importance of verifying the specificity of any
potential biomarker. Regarding the number
of CpG motifs to cover in a biomarker-​based
assay, some publications advocate exploiting
the regional nature of DNA methylation
by measuring the methylation of several
neighbouring CpG repeats using a single
assay41, although the findings of other
studies19 demonstrate that methylation of a
single CpG dinucleotide can be responsible
for transcriptional repression as well as
determining the clinical relevance of a
biomarker. This observation emphasizes the
importance of identifying not only the
optimal genomic location but also
the optimal number of CpG dinucleotides
to be analysed by each individual DNA
methylation-​based biomarker assay.
In order to investigate whether
our strategy of measuring CpG
hypermethylation in the promoter regions
of downregulated genes is the best possible
method of determining the clinical
relevance of DNA methylation, we explored
the hypothesis that the 14 cancer DNA
methylation-​based biomarkers that currently
have commercially available detection assays
(Table 1) are indeed located in the most
clinically relevant genomic region or regions.
Testing this hypothesis is straightforward
owing to the universal availability of
large-​scale cancer genomics databases.
Biomarker performance in TCGA
Publicly available databases such as The
Cancer Genome Atlas (TCGA) already have
demonstrated value for the cancer research
community and have proved helpful in
unravelling both the genetic and epigenetic
background of cancer (see, for example, the
comprehensive molecular classifications of
colorectal42 and prostate43 cancers). Data
provided by TCGA are publicly available
and can be used in biomarker research to
generate hypotheses or as a discovery or
validation data set. Several online tools, such
as the UCSC Xena browser44, cBioPortal45,
and MEXPRESS46, have enabled researchers
to easily inspect (and analyse) TCGA data
for their biomarker of interest.
We used TCGA data to examine the
14 methylation-​based biomarkers with
commercially available assays (Table 1). A
similar analysis of potential biomarkers
that failed to reach the clinic could provide
valuable insight into the pitfalls of the
biomarker development process, but given
the considerable technical challenges such
an analysis would pose, we decided to focus
on the small set of biomarkers for which
assays are commercially available. When
evaluating the genomic location examined
using the ConfirmMDx test to measure
the methylation status of GSTP1, together
with that of regions interrogated by other
primers with published data indicating
their use in prostate adenocarcinoma
samples (Fig. 2; Supplementary Table 3),
all assays were found to target the same
genomic region surrounding the TSS,
despite the existence of small differences
in the exact genomic location analysed.
Each Infinium 450k probe within the
plotted genomic window revealed a clear
difference in methylation between tumour
and nonmalignant tissue samples, as well
as a strong negative correlation with GSTP1
expression levels, indicating the presence of
a 1-kb-​wide region with clinical relevance,
which is targeted by all illustrated markers.
Overall, eight assays examine methylation
of biomarkers located in the canonical
promoter regions of their corresponding
genes (GSTP1, RASSF1, NDRG4, BMP3,
TWIST1, MGMT, BCAT1, and IKZF1),
12 assays examine biomarkers located
within 2 kbp of the TSS (GSTP1, RASSF1,
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 PerspeCTIves

Methylation location of a specific probe (β-​value ≥ 0.2)

Correlation
GSTP1 1 1
0.5
0 0.5
–0.5
–1
GSTP1
1 marker (Supplementary Fig. 3).
4
2 3
6 5
7
9 8
67351000 67351500 67352000
Genomic location
Genomic Spearman
Samples annotation correlation
Normal Gene P < 0.05
Tumour Transcript P ≥ 0.05
Primer
CpG(i)
Fig. 2 | genomic location of GSTP1 Dna methylation biomarkers. All GSTP1 methylation-​based
biomarkers in this figure cluster together at the canonical transcription start site (note that for the
ConfirmMDx assay , only information on the amplicon was available; all other primer annotations can
be found in Supplementary Table 3). On inspection of The Cancer Genome Atlas (TCGA) DNA meth-
ylation and gene expression data for GSTP1 in prostate adenocarcinoma samples, we can see that all
displayed Infinium 450k probes reveal differences in methylation between nonmalignant and tumour
tissue samples (as assessed by Wilcoxon test, false discovery rate (FDR) < 0.05), and that the methyla-
tion data of each probe were negatively correlated with GSTP1 expression (as assessed by Spearman
correlation, rs < −0.5, FDR < 0.05). The y-​axis on the right shows the β-​values; a horizontal bar was drawn
at the median β-​value for each probe, and the vertical lines indicate the 25–75% interquartile range.
The y-​axis on the left shows rs. Hypermethylation of the CpG island (CpG(i)) in the promoter of GSTP1
is a prime example of a DNA methylation-​based biomarker that is associated with downregulation of
the corresponding gene in cancer. Details on the primer sequences were sourced from the following
publications: 1 (ref.57); 2 (refs58,59); 3 (refs60–70); 4 (ref.71); 5 (ref.72); 6 (ref.73); 7 (ref.74); 8 (ref.75);
and 9 (ref.76).
NDRG4, BMP3, TWIST1, MGMT, BCAT1, 2, 4–12), the only exception being the
IKZF1, APC, both SEPT9 markers, and predictive MGMT marker, for which
SHOX2), and all but one of these biomarkers the comparison of nonmalignant and
(APC) are located in a CpG island (Table 1; tumour tissue samples is limited by a lack
Supplementary Figs 1–12). of suitable nonmalignant tissue samples.
In an exploratory analysis, we then Ideally, we would have compared the
considered whether the biomarkers MGMT methylation profiles of patients
examined using commercially available who responded to treatment with
assays were hypermethylated in tumour temozolomide with those of patients
samples and whether their methylation who did not respond to this agent, in
status correlated with the expression of line with the hypothesized effects of
the associated gene by analysing the MGMT methylation on responsiveness
specific probes used at and around to alkylating agents, although data on
the location of each biomarker. We found these responses are not readily available in
a statistically significant difference in TCGA. Instead, we performed a survival
methylation between nonmalignant analysis on TCGA data for all Infinium
and tumour samples at the location of 450k probes surrounding the location
each marker (Wilcoxon test, Benjamini of the MGMT marker, in which we
& Hochberg false discovery rate compared the overall survival outcomes
(FDR) < 0.05) (Supplementary Figs 1, of patients who had methylation at the
(β-value) and those who did not (β-​value < 0.2).
Only data from patients who received
0.75 temozolomide were included in this
analysis. We observed a significant
difference in overall survival at three probe
locations (log-​rank test, FDR < 0.05), one
0.25 of which corresponded with the location
covered by the commercially available
0
MGMT assay, thereby confirming the
known predictive power of MGMT as a
Of the 14 biomarkers, 10 (GSTP1,
NDRG4, BMP3, SHOX2, TWIST1,
OTX1, ONECUT2, MGMT, BCAT1,
and IKZF1) are located in a region
where we found a statistically significant
correlation between methylation and
gene expression by analysing TCGA
Wilcoxon data (Spearman correlation, FDR < 0.05).
test Interestingly, for methylation of SHOX2,
P < 0.05 OTX1, and ONECUT2, this correlation
P ≥ 0.05 was positive instead of negative. Several
investigators describe the complex, but
often positive, relationships between gene-​
body methylation and gene expression in
general47, and a positive association has
been reported for methylation of SHOX2
specifically48. Overall, our findings, while
not definitive evidence, imply that our
proposed strategy is indeed a valid approach
to identifying clinically relevant regions.
TCGA data come with two important
limitations. First, DNA methylation is
measured using Infinium 450k microarrays,
restricting the resulting data to the particular
genomic locations of the probes used in the
array, which might not necessarily capture
the most relevant methylation sites. Second,
for predictive markers, which are associated
with a specific treatment outcome, relevant
data on both treatment received and patient
outcomes are not available in TCGA. Despite
these limitations, data from TCGA or other
large-​scale cancer genomics projects might
still provide valuable information on, for
example, the presence of DNA methylation
or a possible link between DNA methylation
and gene expression. Ongoing efforts by
international organizations, such as the
International Cancer Genome Consortium
(ICGC), to create comprehensive cancer
genomics databases might help to resolve
these issues.
Importantly, none of the standard
bisulfite-​based technologies, including
the Infinium microarrays, can distinguish
between DNA methylation and DNA
hydroxymethylation, another epigenetic
mark linked with tumorigenesis49, unless
a specific protocol is used to identify
hydroxymethylation50. Moreover, other

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Box 1 | Recommendations
• The optimal genomic location of a potential DNA methylation biomarker and the number of
CpGs to cover should be critically evaluated. The location of a DNA methylation-​based
biomarker can affect clinical relevance.
• Publicly available, large-​scale cancer (epi)genomics databases should be used to identify the
genomic location that is best associated with the relevant clinical features.
• All assay protocols should be accurately and thoroughly reported, including the exact genomic
location of the biomarker.
approaches to finding clinically relevant
changes in DNA methylation are available,
such as the location-​agnostic machine
learning methods used in the EPICUP
classifier, which was developed to identify
and classify the origins of cancers of
unknown primary51.
Reporting
The clinical translation of a methylation-​
based cancer biomarker does not depend
on identifying the most clinically relevant
genomic location alone: for observations
regarding genomic location to be useful
they must also be published at a sufficient
level of detail. Reproducibility, one of the
pillars of scientific research, relies on the
detailed and transparent reporting of all
findings, data, and protocols, including
the genomic location of methylation
biomarkers10,52,53. Accurate reporting seems
obvious but, in practice, improvements are
still possible52. In our opinion, details of the
genomic locations of all methylation-​based
biomarkers with commercially available
assays were reported in sufficient detail,
apart from GSTP1, APC, and RASSF1, for
which only the genomic location of the
MSP amplicon was published and not the
primer sequences. We recommend that, in
future, publications contain details such
as specific primer sequences and assay
protocols, preferably in the main text of
the article (and not as supplementary
information), to facilitate the replication of
the findings. Reporting only the genomic
location of the primers or amplicons and/
or referencing previously published primers
without providing the exact sequences
either burdens the interested reader with
the additional task of retrieving the primer
sequences or necessitates the design of
new assays, which might differ from those
used in the original publication. Finally,
having to contact the authors to obtain
primer sequences or assay protocols
only results in further complications
in terms of both data exchange and in
the reproducibility of research — two
cornerstones of good science. A reporting
standard for cancer DNA methylation-​based
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
biomarkers comparable to, for example,
the Minimum Information About a
Microarray Experiment standard
for microarray experiments54 and based
on the nomenclature proposed by the
Human Genome Variation Society55,56
could help alleviate these issues.
Recommendations
In summary, the analysis of the
commercially available assays designed
to quantify various methylation-​based
biomarkers, together with the suboptimal
assay design regarding genomic location
regularly encountered in the literature,
prompted us to establish a few simple
recommendations that will hopefully
facilitate the development and clinical
translation of DNA methylation-​based
markers (Box 1). Our first and most
important recommendation is to critically
evaluate both the optimal genomic
location of a potential methylation-​
based biomarker and the number of
CpG dinucleotides to cover in the assay
used to measure DNA methylation,
regardless of whether this biomarker
is located in a promoter CpG island or
in another functional region of a gene.
The examples described above provide
overwhelming evidence that the location
of a DNA methylation-​based biomarker
absolutely influences its clinical relevance.
Second, we recommend incorporating
publicly available large-​scale epigenomics
data into methylation-​based biomarker
development and validation processes
in order to improve predictions of the
genomic region that is best associated
with the relevant clinical features.
Third, in order to facilitate independent
replication and validation, we urge all
investigators to accurately report the
methylation-​based biomarker assay
protocols used, including details of the
exact genomic location of the biomarker.
Other steps in the development process,
such as the choice and design of an
appropriate assay, comparisons of the
performance of any new biomarker to
the gold standard (if available), and/or
demonstration of its clinical utility, are
equally important but are not the focus of
these recommendations.
Conclusions
DNA methylation-​based biomarkers were
heralded as the next ‘big thing’ in cancer
biomarker research more than a decade
ago, but so far, they have struggled to live
up to the expectations. Several possible
explanations are available as to why so few
DNA methylation-​based biomarkers have
been commercialized, but we believe that
the complex relationship between DNA
methylation and its precise genomic location
is one of the main obstacles. In order to
reduce the difference between the number of
published biomarkers and the number
of biomarkers that are used in the clinic, we
formulated three simple recommendations
for the development of DNA methylation-​
based biomarkers. Biomarkers developed
according to these recommendations are
not guaranteed to be either reproducible
or clinically relevant, although, at least
the first barrier in the DNA methylation-​
based biomarker pipeline, determining
the clinically most relevant region, can
be overcome though analysis of publicly
available data. In order to systematically
evaluate the performance of DNA
methylation-​based biomarkers and to
support the accurate future reporting
of both the discovery and validation of
such biomarkers, we are developing a
centralized atlas containing data on all
published biomarkers, as well as a biomarker
reporting standard. We hope that our
recommendations and continuing efforts
will help improve the performance of DNA
methylation-​based markers, promote
reproducibility, and decrease the amount of
research waste that is currently produced in
this field.
Alexander Koch   1, Sophie C. Joosten2, Zheng Feng1,
Tim C. de Ruijter2, Muriel X. Draht1, Veerle Melotte1,
Kim M. Smits1,2, Jurgen Veeck2, James G. Herman3,
Leander Van Neste1, Wim Van Criekinge4,
Tim De Meyer5 and Manon van Engeland1*
1
Department of Pathology, GROW — School for
Oncology and Developmental Biology, Maastricht
University Medical Center, Maastricht, Netherlands.
2
Division of Medical Oncology, Department of Internal
Medicine, GROW — School for Oncology and
Developmental Biology, Maastricht University Medical
Center, Maastricht, Netherlands.
3
Lung Cancer Program, University of Pittsburgh Cancer
Institute, The Hillman Cancer Center, Pittsburgh,
PA, USA.
4
BioBix: Laboratory of Bioinformatics and
Computational Genomics, Department of Mathematical
Modelling, Statistics and Bioinformatics, Ghent
University, Ghent, Belgium.
www.nature.com/nrclinonc

5
Department of Mathematical Modelling, Statistics
and Bioinformatics, Ghent University, Ghent, Belgium.
*e-​mail: manon.van.engeland@mumc.nl
https://doi.org/10.1038/s41571-018-0004-4
Published online xx xx xxxx
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Acknowledgements
The work of the authors is supported financially by the Maag
Lever Darm Stichting (MLDS, grant FP13-15) and an SU2C-​
DCS International Translational Cancer Research Dream
Team Grant (Stand Up To Cancer (SU2C)-AACR-​DT1415,
MEDOCC). SU2C is a programme of the Entertainment
Industry Foundation administered by the American
Association for Cancer Research and the Universiteitsfonds
Limburg/SWOL.
Author contributions
A.K., S.C.J., and M.v.E. researched data for the article, all
authors made a substantial contribution to discussion of con-
tent, A.K., S.C.J., and M.v.E. wrote the manuscript, and all
authors reviewed and/or edited the manuscript before
submission.
Competing interests
M.v.E. receives research funding from MDxHealth. W.V.C. is
a consultant of MDxHealth. The other authors declare no
competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
Supplementary information
Supplementary information is available for this paper at
https://doi.org/10.1038/s41571-018-0004-4.
Related links
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http://icgc.org/
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