Interpretation of genome-wide infinium methylation data from ligated DNA in formalin-fixed, paraffin-embedded paired tumor and normal tissue.

Abstract

Formalin-fixed, paraffin-embedded (FFPE) samples are a highly desirable resource for epigenetic studies, but there is no suitable platform to assay genome-wide methylation in these widely available resources. Recently, Thirlwell et al. (2010) have reported a modified ligation-based DNA repair protocol to prepare FFPE DNA for the Infinium methylation assay. In this study, we have tested the accuracy of methylation data obtained with this modification by comparing paired fresh-frozen (FF) and FFPE colon tissue (normal and tumor) from colorectal cancer patients. We report locus-specific correlation and concordance of tumor-specific differentially methylated loci (DML), both of which were not previously assessed.
We used Illumina's Infinium Methylation 27K chip for 12 pairs of FF and 12 pairs of FFPE tissue from tumor and surrounding healthy tissue from the resected colon of the same individual, after repairing the FFPE DNA using Thirlwell's modified protocol.
For both tumor and normal tissue, overall correlation of β values between all loci in paired FF and FFPE was comparable to previous studies. Tissue storage type (FF or FFPE) was found to be the most significant source of variation rather than tissue type (normal or tumor). We found a large number of DML between FF and FFPE DNA. Using ANOVA, we also identified DML in tumor compared to normal tissue in both FF and FFPE samples, and out of the top 50 loci in both groups only 7 were common, indicating poor concordance. Likewise, while looking at the correlation of individual loci between FFPE and FF across the patients, less than 10% of loci showed strong correlation (r ≥ 0.6). Finally, we checked the effect of the ligation-based modification on the Infinium chemistry for SNP genotyping on an independent set of samples, which also showed poor performance.
Ligation of FFPE DNA prior to the Infinium genome-wide methylation assay may detect a reasonable number of loci, but the numbers of detected loci are much fewer than in FF samples. More importantly, the concordance of DML detected between FF and FFPE DNA is suboptimal, and DML from FFPE tissues should be interpreted with great caution.

Full text

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RESEARCH ARTICLEOpen Access
Interpretation of genome-wide infinium
methylation data from ligated DNA in formalin-
fixed, paraffin-embedded paired tumor and
normal tissue
Farzana Jasmine1, Ronald Rahaman1, Shantanu Roy1, Maruf Raza6, Rupash Paul6, Muhammad Rakibuz-Zaman5,
Rachelle Paul-Brutus1, Charlotte Dodsworth1, Mohammed Kamal6, Habibul Ahsan1,2,3,4and Muhammad G Kibriya1*
Abstract
Background: Formalin-fixed, paraffin-embedded (FFPE) samples are a highly desirable resource for epigenetic
studies, but there is no suitable platform to assay genome-wide methylation in these widely available resources.
Recently, Thirlwell et al. (2010) have reported a modified ligation-based DNA repair protocol to prepare FFPE DNA
for the Infinium methylation assay. In this study, we have tested the accuracy of methylation data obtained with
this modification by comparing paired fresh-frozen (FF) and FFPE colon tissue (normal and tumor) from colorectal
cancer patients. We report locus-specific correlation and concordance of tumor-specific differentially methylated
loci (DML), both of which were not previously assessed.
Methods: We used Illumina’s Infinium Methylation 27K chip for 12 pairs of FF and 12 pairs of FFPE tissue from
tumor and surrounding healthy tissue from the resected colon of the same individual, after repairing the FFPE DNA
using Thirlwell’s modified protocol.
Results: For both tumor and normal tissue, overall correlation of b values between all loci in paired FF and
FFPE was comparable to previous studies. Tissue storage type (FF or FFPE) was found to be the most significant
source of variation rather than tissue type (normal or tumor). We found a large number of DML between FF
and FFPE DNA. Using ANOVA, we also identified DML in tumor compared to normal tissue in both FF and FFPE
samples, and out of the top 50 loci in both groups only 7 were common, indicating poor concordance.
Likewise, while looking at the correlation of individual loci between FFPE and FF across the patients, less than
10% of loci showed strong correlation (r ≥ 0.6). Finally, we checked the effect of the ligation-based modification
on the Infinium chemistry for SNP genotyping on an independent set of samples, which also showed poor
performance.
Conclusion: Ligation of FFPE DNA prior to the Infinium genome-wide methylation assay may detect a reasonable
number of loci, but the numbers of detected loci are much fewer than in FF samples. More importantly, the
concordance of DML detected between FF and FFPE DNA is suboptimal, and DML from FFPE tissues should be
interpreted with great caution.
* Correspondence: kibriya@uchicago.edu
1Department of Health Studies, The University of Chicago, Chicago, IL 60637,
USA
Full list of author information is available at the end of the article
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© 2012 Jasmine et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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Background
Aberrant DNA methylation is a well-established path-
way in carcinogenesis [1,2]. In colorectal cancer (CRC),
global hypomethylation of DNA and gene-specific
hypermethylation of tumor suppressor genes and micro-
RNA genes are extensively studied [3]. For example,
seminal work in cancer epigenetics has shown that most
cases of microsatellite-instable CRC are caused by the
hypermethylation and consequent silencing of the mis-
match-repair gene MLH1 [4,5]. Many epigenetic mar-
kers for CRC are now known, including MGMT [6,7],
VIM [8], APC [9], RUNX3 [5,10], CDKN2A [11], and
numerous others found in recent genome-wide studies
[12-14]. It is hoped that continuing studies can provide
useful strategies for detection, treatment, and the under-
standing of etiology [15].
Formalin-fixed, paraffin-embedded (FFPE) samples are
routinely collected for histopathological diagnosis and
are thus a highly desirable resource for epigenetic stu-
dies. Though formalin fixation does not alter the methy-
lation status of cytosine [16], it does cause other forms
of DNA damage, including cross-linking, fragmentation,
and generation of apurinic/apyrimidinic sites [17]. This
degradation can be detrimental to qPCR [18] or whole-
genome amplification (WGA) [19], which are integral
steps in many methylation assays.
Therefore, any existing methylation assay must be
carefully evaluated before it can be confidently used for
FFPE-derived DNA. Many methylation assays have been
evaluated for such purposes [20-26]. The most compre-
hensive validations involve comparisons between paired
FFPE and fresh-frozen (FF) tissue samples, such as the
validations reported for: high-resolution melting analysis
[27], qPCR quantification after methylation-specific
restriction enzyme digestion [20], bisulfite sequencing
[21], and Illumina’s GoldenGate methylation assay [23].
Killian’s validation of the GoldenGate assay showed
good correlation between paired FFPE and FF samples
but the GoldenGate assay interrogates a limited number
of CpG loci and is not suitable for studies of large num-
bers of loci [23]. For genome-wide studies, Illumina’s
Infinium assay [28] allows thousands of loci to be inter-
rogated at a time. However, the Infinium chemistry
depends on WGA and thus was originally designed for
high-quality, high molecular weight DNA. Many WGA
protocols fail with fragmented FFPE DNA [19], and Illu-
mina’s proprietary Infinium WGA chemistry has been
shown to fail with these samples by both in-house [29]
and independent results [30]. GoldenGate chemistry, on
the other hand, does not involve WGA, targets small
fragments of DNA for PCR amplification, and thus may
be compatible with FFPE DNA. In a previous study we
comparedgenome-widegeneexpression(using
Illumina’s DASL assay, based on GoldenGate chemistry)
in paired FF and FFPE breast tumor tissues and sur-
rounding healthy tissues [31]. In that study, we found
that the tumor specific differentially expressed genes
detected in FF and FFPE samples were significantly dif-
ferent, suggesting that interpreting FFPE gene expres-
sion data may be problematic.
Recently, Thirlwell et al. described a modified Infi-
nium methylation protocol in which FFPE DNA was
repaired by ligation prior to the bisulfite conversion and
methylation assay [30]. Thirlwell’s protocol was shown
to be effective in several respects. First, the authors
showed that ligation allowed successful WGA, whereas
unligated replicates failed to amplify. Second, the
authors demonstrated reasonable correlation between
paired FF and FFPE samples from primary ovarian can-
cer tissues. However, they did not report whether FFPE
tumor samples could detect the same differentially
methylated loci (DML) as were detected by examining
FF tumor tissue. Recently, we reported results from a
genome-wide DNA methylation study in colorectal can-
cer using Illumina’s Infinium-based HumanMethyla-
tion27 microarray [12]. The study was conducted using
paired FF tumor and adjacent normal colon tissue sam-
ples from 24 patients. FFPE tumor and normal samples
were also available from the same patients. This pro-
vided an excellent opportunity to independently test the
utility of ligated-FFPE DNA for Infinium methylation
analysis using Thirlwell’s modification [30]. In the cur-
rent study, we have tested the accuracy of methylation
data from ligated-FFPE DNA through numerous correla-
tions with paired FF DNA for both CRC and adjacent
normal colon tissue. We also identified DML in FF
tumor DNA (compared to FF adjacent normal) and
compared these to loci that were differentially methy-
lated in ligated-FFPE tumor DNA (compared to ligated-
FFPE adjacent normal) from the same patients. Our
study is unique amongst other validations of FFPE
methylation assays in that we have reported results for
locus-specific correlations across samples and concor-
dance of tumor-specific DML.
Methods
Tissue samples
Colon tissues (tumor and surrounding healthy) were
collected from surgically removed colonic segments
from consecutive patients at Bangabandhu Sheikh Mujib
Medical University (BSMMU), Dhaka, Bangladesh, as
described previously [12]. All samples were collected by
one surgical pathology fellow (MR) from the operating
room immediately after surgical resection during the
period of December 2009 to March 2010. Histopathol-
ogy was done independently by two histopathologists
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(MK & MR), and there was concordance in all cases.
For each patient, one sample was collected from the
tumor mass, and another sample was taken from the
resected, unaffected part of the colon about 5-10 cm
away from the tumor mass. From each site, tissue sec-
tions were preserved as (1) fresh frozen, (2) in RNA-sta-
bilizing buffer and (3) as FFPE block. The samples were
shipped on dry ice to the molecular genomics lab at the
University of Chicago for subsequent DNA extraction
and methylation assay. We also received the correspond-
ing FFPE blocks that were used for histopathology.
Written informed consent was obtained from all partici-
pants. The research protocol was approved by the “Ethi-
cal Review Committee, Bangabandhu Sheikh Mujib
Medical University”, Dhaka, Bangladesh (BSMMU/2010/
10096) and by the “Biological Sciences Division, Univer-
sity of Chicago Hospital Institutional Review Board”,
Chicago, IL, USA (10-264-E). We have previously
reported genome-wide methylation data from the first
24 paired (tumor and corresponding healthy colonic tis-
sue) FF DNA [12]. In this paper we present methylation
data from FFPE sections of the first 12 consecutive
patients of the same series for whom we had paired
(normal and CRC) FFPE blocks available and compared
the data with corresponding 12 pairs from DNA from
FF samples.
DNA extraction and quality control
DNA was extracted from FFPE tissue (tumor and sur-
rounding healthy tissue) using the Puregene Core kit A
(Qiagen, Maryland, USA). During extraction all DNA
samples were treated with RNase. FFPE tissues were
about 1 year old. All DNA concentrations were mea-
sured by Nanodrop (Thermo-Fisher, USA), and integrity
was checked by the Agilent Bioanalyzer 2100 using the
DNA 12000 kit (Agilent Technologies, USA).
Bisulfite conversion
2 μg FFPE DNA were ligated before starting bisulfite
conversion using the protocol described by Thirlwell et
al. [30]. For bisulfite conversion, the EZ DNA methyla-
tion kit (Zymo Research, USA) was used.
Genome-wide methylation assay
The Infinium Methylation assay (Illumina Inc., USA)
was done using the Methylation 27K chip, which con-
tains 27,578 CpG sites spanning 14,495 genes. The CpG
sites were located within the proximal promoter regions
of genes, with the distance to transcription start site
(TSS) ranging from 0 to 1499 bp and averaged at 389 ±
341 bp. Paired FFPE DNA from CRC and surrounding
normal colonic tissues were processed on the same chip
to avoid batch effects, and all 24 FFPE samples were
processed on 2 chips (12 samples per chip). It may be
noted that the corresponding 24 FF samples were pro-
cessed in a different batch previously, but the corre-
sponding DNA samples from normal and CRC tissue
were processed in the same chip. A Tecan Evo robot
was used for automated sample processing and the
chips were scanned on a single BeadArray reader (S428).
Illumina’s BeadStudio analytical software showed excel-
lent intensity for staining (above 15000), clear clustering
for the hybridization probes, good target removal inten-
sity (< 400) and satisfactory bisulfite conversion.
Genome-wide methylation data analysis
For measuring methylation, we used the Illumina Bead-
Studio software to generate the b value for each locus
from the intensity of methylated and unmethylated
probes. The b value is calculated as (intensity of methy-
lated probe)/(intensity of methylated probe + intensity
of unmethylated probe). Hence, b ranges between 0
(least methylated) and 1 (most methylated) and is pro-
portional to the degree of the methylated state of a par-
ticular loci. The methylation module of BeadStudio was
used for differential methylation analysis using Illumi-
na’s custom model. The model operates under the
assumption that the methylation value b is normally dis-
tributed among biological replicates corresponding to a
set of biological conditions (tumor and normal in the
present scenario). The DiffScore of a probe is computed
as:
DiffScore = 10Sign(βtumor− βnormal)log10p
where p represents the p-value from t-test.
Deltaβ = (βtumor− βnormal)
In addition to the BeadStudio differential methylation
analysis, we exported the BeadStudio generated b values
to the PARTEK Genomic Suite [32] for further statisti-
cal analyses. Principal component analysis (PCA) and
sample histograms were checked as a part of quality
control analyses of the data. Mixed-model multi-way
ANOVA (which allows more than one ANOVA factor
to be entered in each model) was used to compare the
individual CpG loci methylation data across different
groups. Two of the FFPE samples were excluded from
the analysis due to poor gene detection. The remaining
analyses were done with 10 pairs of FF and FFPE tissue.
In general, tissue type (tumor or adjacent normal), sex
(male or female) and tumor location (proximal colon or
distal colon) were used as categorical variables with
fixed effect since these represent all conditions of inter-
est; whereas “case ID#” (used as a proxy of inter-person
variation) was treated as a categorical variable with ran-
dom effect, since the person ID is only a random sample
of all the levels of that factor. Method of moments
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estimation was used to obtain estimates of variance
components for mixed models [33]. In the ANOVA
model, the b value for a locus was used as the response
variable, and tissue type (tumor or normal), age category
(≤ 40 yrs vs. > 40 yrs), case ID#, sex and location were
entered as ANOVA factors. It may be noted that age
category, sex and location were nested within case ID#.
One example of a model is as follows:
Yijklmn= μ+Tissuei+Age Cat40j+Sexk+Location1+Person(Age Cat40 ∗ Sex ∗ Location)jklm+εijklmn
Where Yijklmnrepresents the nthobservation on the ith
Tissue, jthAge_Cat40, kthSex, lthLocation and mthPer-
son; μ is the common effect for the whole experiment,
εijklmnrepresents the random error present in the nth
observation on the ithTissue, jthAge_Cat40, kthSex, lth
Location, and mthPerson. The errors εijklmnare assumed
to be normally and independently distributed with mean
0 and standard deviation δ for all measurements. An
FDR of 0.05 was used for multiple testing correction.
The correlation of b values between FF and FFPE
samples from each individual was checked in both nor-
mal and tumor tissue. Then the correlation of the aver-
age b values of all the FF and corresponding FFPE
samples was analyzed both for normal and tumor tissue.
The distribution of the genes correlated between FF
and FFPE samples were also checked. The top 50 differ-
entially methylated genes between normal and tumor
tissue in both FF and FFPE tissue were detected to find
the common genes.
Results
Detection of loci
In the microarray, a locus was said to be detected if the
average signal intensity of that locus was significantly (p
< 0.05) higher than the built-in negative control on the
chip. In terms of the number of detected loci per sam-
ple, there was no statistical difference between normal
and tumor DNA from FF tissue. However, in FFPE
DNA, for both normal and tumor tissues, the number
of detected loci per sample was significantly lower than
in FF DNA (Figure 1), although there was no difference
in the number of detected loci between normal and
tumor within FFPE blocks. The histogram showing the
distribution of signal intensity is presented in Additional
file 1: Figure S1. The data discussed in the publication is
deposited in NCBI’s Gene Expression Omnibus and will
be accessible through GEO (accession #GSE33181).
Sources of variation in methylation
Principal components analysis (Figure 2) shows that the
samples cluster by both storage type (FF vs. FFPE) and
tissue type (tumor vs. normal). There is much greater
separation between samples of different storage type
(PC1 = 33.3%, shown on the x-axis) than between sam-
ples of different tissue type (PC2 = 10.4%, shown on the
y-axis). It is also notable that, for FFPE tissue, the clus-
tering of tumor and normal samples shows poorer
separation than the clustering of tumor and normal
samples in FF tissue.
Histograms of methylation b-values by storage type
The distribution of b values for FF (Figure 3, shown in
red) and FFPE samples (shown in blue) clearly shows
that the vast majority of loci were hypomethylated
(below 0.15) and only a few were hypermethylated
(above 0.5) in all samples. However, the FFPE samples
clustered slightly separately than FF samples.
Correlation between storage-type and tissue-type pairs
b values of all 27,578 loci from each FFPE sample were
plotted against the b values from corresponding FF tis-
sue of the same patient. Representative scatter plots
from one patient (C_1) are shown in Figure 4A (for nor-
mal tissue) and 4B (for tumor tissue), where each dot
Figure 1 Number of loci detected in each sample group. The
sample groups shown are: tumor FF tissue (red, average detected
loci = 27530.6 ± 27.4); tumor FFPE tissue (blue, 27,239.0 ± 375.4),
normal FF tissue (green, 27,541.1 ± 26.7), and normal FF tissue
(violet, 27,102.5 ± 502.0). There was significant difference between
average detected loci in normal FF vs. normal FFPE (p = 0.002) and
between tumor FF vs. tumor FFPE (p = 0.032).
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represents a locus in a single sample. Overall the degree
of scatter suggests that, at the individual patient level,
the methylation status in FFPE tissue correlated poorly
with paired FF tissue. This poor correlation may be
explained by the DNA damage from formalin fixation. It
may be noted that we have seen technical replicates to
show very tight correlation (r2> 0.99) in the Infinium
methylation assay (see Additional file 2: Figure S2).
Then the b values of all 27,578 loci from each tumor
sample were plotted against the b values from the corre-
sponding normal tissue of the same patient. Representa-
tive scatter plots from one patient (C_1) are shown in
Figure 4C (for FF) and 4D (for FFPE). As expected, the
scatter plots indicate differential methylation of a num-
ber of loci in CRC tissue compared to normal tissue.
In the next step, instead of using b values from indivi-
dual patients, we used the mean b values from all 10
patients and plotted the values from FFPE against FF.
Scatter plots showed that the correlation calculated in
the total samples was better (r2= 0.89 for normal tissue
and r2= 0.93 for tumor tissue) than the correlation that
was seen at the individual sample level (Figure 5A and
5B). Even then, the scatter suggests that many loci do
not correlate well between FF and FFPE.
Similarly, we looked at the correlation of mean b
values of tumor FF tissue vs. mean b-values of normal
FF tissue (r2= 0.93, Figure 5C); and mean b values of
tumor FFPE tissue vs. mean b values of normal FFPE
tissue (r2= 0.91, Figure 5D). Average tumor vs. normal
b values showed better correlation than individual
Figure 2 Principle components analysis, displaying spatial
separation of: FF tumor tissue (red), FF normal tissue (green),
FFPE tumor tissue (blue), and FFPE normal tissue (purple). The
color coding suggests that the separation along the horizontal axis
(PC1) may be attributed to storage type (FF vs. FFPE), and the
separation along the vertical axis (PC2) may be attributed to tissue
type (tumor vs. normal).
Figure 3 Histograms of methylation b-values by storage type.
The histograms show the frequency distribution of 26,486
autosomal loci in FF samples (red) or FFPE samples (blue), where
each line represents one sample.
Figure 4 Scatter plots of b-values for sample pairs from
patient C1. Each point represents the b value of one locus in this
patient. For each plot, the straight line is the regression line and r2
is Pearson’s correlation coefficient squared. Top: Correlation
between paired FF and FFPE samples for (A) normal colon tissue
and (B) tumor tissue. Bottom: Correlation between paired tumor
and normal samples for (C) FF tissue and (D) FFPE tissue.
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sample pairs, for both FF and FFPE tissue. This increase
in r2is related to the direct increase in data points used
for analysis.
Locus-specific correlation between FF and FFPE DNA
Methylation is significantly different between males and
females, mainly due to the sex chromosomal loci [12];
therefore we excluded the sex chromosomal loci from
subsequent analyses. For each of the 26,486 autosomal
loci, we correlated b values of FF and FFPE in all 20
paired samples (Figure 6). Representative scatter plots
for two individual loci are shown in Figure 6A and 6B,
where each dot represents a sample. The distribution of
the resultant r values for all 26,486 loci are shown in
the histogram in Figure 6C. The figure clearly shows
that less than 10% of the loci correlated well in FF and
FFPE samples (only 2,463 loci had r ≥ 0.6), and the vast
majority of the loci showed poor correlation between
corresponding FF and FFPE tissue. Therefore, our data
suggests that the use and interpretation of the correla-
tion analyses requires caution, especially when consider-
ing a large number of loci per sample.
Differentially methylated loci (DML) in FFPE compared to
FF
In a paired t-test, there were a total of 17,896 autosomal
loci (67.56%) that were significantly differentially methy-
lated at FDR 0.05; of these, the absolute Δb was greater
than 0.2 for 922 loci. In an unpaired t-test with boot-
strapping, 9,475 autosomal loci (35.77%) were differen-
tially methylated (bootstrap ≤ 0.05); of these, the
absolute Δb was greater than 0.2 in 652 loci. We also
used a multivariate ANOVA model that controls for tis-
sue type (tumor vs. normal), person-to-person variation,
age category (above or below 40 yrs), sex, tumor loca-
tion (proximal vs. distal); this analysis also revealed dif-
ferential methylation in a total of 18,660 loci (70.45%) at
FDR 0.05 level that were differentially methylated in
FFPE tissue compared to corresponding FF tissue. Of
these, the absolute Δb was greater than 0.2 in 914 loci.
Overlap between these analyses is presented in Addi-
tional file 3: Figure S3, which clearly indicates that
regardless of which statistical test is applied, there are a
large number of loci that show significant differential
methylation in FFPE samples compared to correspond-
ing FF sample. Unsupervised clustering using these loci
can very effectively differentiate FFPE samples from FF
samples (Figure 7).
Differentially methylated loci in tumor tissue
In the next step, we examined whether comparing FFPE
tumor samples against FFPE normal samples generates a
list of tumor-specific DML that is similar to the list gen-
erated from FF tumor and FF normal tissue. In FF sam-
ples we identified the DML in CRC using ANOVA after
controlling for person-to-person variation, tumor loca-
tion (left or right colon), sex, and age. In this analysis, a
total of 1,404 loci were differentially methylated at p ≤
0.05 level and Δb > 0.2; among these loci, 590 passed
the criteria of FDR 0.05 and Δb > 0.2. Hierarchical clus-
tering using the top 50 of these DML (covering 46
Figure 6 Correlation between FF and FFPE b values for
individual methylation loci. For (A) and (B), each data point
represents a single sample for which the b value of the FFPE is
shown on the x-axis and the corresponding b value from FF is
shown on the y-axis. Red points are tumor samples, and blue points
are from normal samples. (A) A representative locus for which
correlation between FF and FFPE samples was poor. (B) A
representative locus for which correlation between FF and FFPE
samples was good. (C) Histogram showing the distribution of
resultant r values from FF/FFPE correlations for all loci.
Figure 5 Scatter plots of average b values for all sample pairs.
Each point represents the b value of one locus averaged over all 10
patients. (A) Average b values of normal FFPE tissue vs. average b
values of normal FF tissue. (B) Average b values of tumor FFPE
tissue vs. average b values of tumor FF tissue. (C) Average b values
of tumor FF tissue vs. average b values of normal FF tissue. (D)
Average b values of tumor FFPE tissue vs. average b values of
normal FFPE tissue.
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genes) is presented in Figure 8A, which shows that the
methylation status of these loci was able to effectively
separate tumor and normal samples. Similarly, using
FFPE (normal and tumor) samples, we also attempted to
identify DML using the same ANOVA method. A total
of 927 loci were differentially methylated at p ≤ 0.05
level and Δb > 0.2, but none of them passed the criteria
of FDR 0.05 and Δb > 0.2. However, for the purposes of
comparison, we selected the top 50 DML in FFPE
(based on unadjusted p-value, covering 40 genes) for
unsupervised hierarchical clustering. It was interesting
to see that even these genes were able to effectively
separate tumor and normal tissue (Figure 8B). When we
compared the top 50 DML from FF and the top 50
DML from FFPE samples, there were only 7 loci com-
mon to both sets covering six genes: EYA4, TFPI2,
GATA4, SPG20, WT1 and SORC53. This overlap is
representative, as the top 100 DML from FF and FFPE
samples had 19 loci in common, and the top 30 DML
from both sets had 2 loci in common (data not shown).
The top 20 DML in FF and FFPE DNA are presented in
Additional file 4: Table S1. We also looked at the corre-
lations of the ANOVA p-values of individual DML from
FFPE and FF samples. The log10-transformed p-values
from FF are shown on the x-axis and those from FFPE
are shown on the y-axis of the Additional file 5: Figure
S4. This also shows lack of strong correlation between
them. We also looked at the correlations of b values
from paired FF and FFPE samples for a few selected
genes e.g. MGMT, MLH1, VIM, APC, and RUNX3,
which are usual suspects in candidate gene approach
studies. In general, the result showed suboptimal corre-
lations (see Additional File 6 for all the 49 probes on
the chip for these five genes). For example, the chip had
Figure 8 Unsupervised clustering and heatmap based on the
top 50 DML in tumor samples compared to paired normal. (A)
The top 50 DML derived from analysis of FF tissue alone. (B) The
top 50 DML derived from analysis of FFPE tissue alone.
Figure 7 Unsupervised clustering and heatmap based on the
top 50 DML that differentiate FF and FFPE samples in
combined analysis of all samples studied.
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a total of 28 loci for MGMT gene and the r2for differ-
ent loci ranged between 0.000001 and 0.29. For MLH1,
however, the r2was up to 0.68.
Next, we compared the sources of variation in the top
50 DML from FF and FFPE sample sets (Figure 9). We
used the same ANOVA model mentioned above, which
factored in person-to-person variation, tissue type
(tumor or normal), tumor location (left or right colon),
sex, and age. For the top 50 DML in FF samples, tissue
type (tumor vs. normal) accounted for a large propor-
tion (70.0%) of the variation in b values. However, for
the top 50 DML in FFPE samples, a smaller proportion
of variation (58.8%) could be attributed to tissue type.
Furthermore, a much larger proportion of variation in
FFPE samples was attributed to person-to-person varia-
tion (19.6% in FFPE vs. 8.7% in FF).
Our data suggests that data generated by Thirlwell’s
modified Infinium methylation protocol can identify >
95% loci in FFPE samples, which is significantly fewer
than what is seen in FF samples in the unmodified Infi-
nium protocol. Based on these data, it is also possible to
separate the tumor and normal samples. In fact, DML
sets from FF and FFPE tissue are both effective at differ-
entiating tumor and normal tissue (Figure 8A and 8B).
However, these DML sets are very discordant, suggest-
ing that using the modified Infinium assay with FFPE
samples may not provide the same biological informa-
tion as the unmodified assay with FF samples (the gold
standard in this case). Furthermore, the DML from
FFPE show a greater amount of variation that cannot be
attributed to differences in tissue type (Figure 9).
Finally, we investigated the effect of the additional
ligation step for FFPE samples in Illumina’s Infinium
genotyping assay, which relies on the same chemistry as
the methylation assay and can provide independent vali-
dation of some of our findings. We compared the per-
formance of the Infinium genotyping assay in ligated
FFPE DNA and unmodified FF DNA, using an indepen-
dent set of 16 DNA samples from 4 patients assayed
with Illumina Human Cyto12 SNP Chips. For each
patient, we had DNA from tumor and surrounding nor-
mal tissue from both FF and FFPE blocks. Ligation was
performed on the FFPE samples prior to WGA, as in
the modified Infinium Methylation assay. Figure 10A
clearly shows that, despite the additional ligation step,
the FFPE samples showed significantly lower call rates
than corresponding FF samples. The logR ratio and the
B-allele frequency plots from representative pairs (Fig-
ures 10B-E) also show higher noise and poor perfor-
mance in ligated FFPE samples. The poor genotype call
rates explain at least part of the poor correlation of
methylation data in FFPE and FF samples. This further
strengthens our conclusion that ligation of FFPE DNA
prior to Infinium processing does not make the Infinium
chemistry fully compatible with FFPE DNA samples.
Discussion
Our study has attempted to validate Thirlwell’s modified
Infinium protocol by using 12 pairs FF and FFPE sam-
ples from 12 primary CRC samples and 12 adjacent nor-
mal tissues. Thirlwell et al. [30] compared 2 FF ovarian
cancer tissue with paired FFPE DNA from ligated and
unligated replicates. The authors showed good correla-
tion of b values and intensity between FF and ligated
FFPE DNA compared to unligated FFPE DNA. How-
ever, Thirlwell did not report changes in differential
methylation that are typically investigated in cancer
research - for example, whether ligated-FFPE DNA pro-
duced the same DML sets as FF DNA, or whether
ligated-FFPE DNA had the same power to distinguish
tumor samples from normal samples. This kind of
hypothesis testing is of particular concern since we have
previously reported that in Illumina’s DASL whole-gen-
ome gene expression microarray, FFPE RNA can yield
significantly different results compared to paired FF
RNA [31].
In our study, the overall correlation of b values were
comparable to Thirlwell’s study [30]. When looking at
the correlation between FF and FFPE biological repli-
cates, Thirlwell found a median r2= 0.91, range = 0.88 -
0.96. This was slightly higher than our observed correla-
tions, but not as high as correlations reported by Killian
for the GoldenGate assay [23], which ranged from r2=
0.95 to 0.99, and it may be noted that GoldenGate
chemistry is suitable for FFPE samples whereas in prin-
ciple Infinium chemistry is not. However, our data sug-
gests that correlation is definitely related to the number
of data points in the analysis.
Ideally, our experiment would have been designed to
include all the four samples from same patient (tumor
and normal from FF and FFPE sections) on the same
chip to totally eliminate any batch effects. Unfortunately,
the FF samples were processed earlier for a separate
study. However, in both the cases (FF and FFPE) paired
tumor-normal samples were processed on the same
chip. Since the b value is calculated from the ratio of
Figure 9 Partial contribution of the different sources of
variation in the methylation data for the top 50 DML derived
from (A) FF tissues and (B) FFPE tissues.
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the signal intensity values (methylated to total), slight
differences in intensities are less likely to affect b.
A few studies have evaluated the use of FFPE tissue
was evaluated for lower throughput methylation assays
with fewer CpG locations. Balic et al. [27] used HRM to
interrogate promoter methylation of two genes (MGMT
and APC), and compared results from paired FFPE and
FF samples in 5 human breast cancer cell lines and 3
human prostate cancer cell lines; these results were also
validated with the MethyLight qPCR assay. Gagnon et
al. validated promoter methylation status of PLAU and
TIMP3 genes in FFPE tissue using methylation sensitive
restriction enzyme digestion and qPCR; this was done
for paired FFPE and FF samples from 9 primary breast
tumor samples and 4 cell line admixtures [20]. Killian et
al. evaluated the GoldenGate methylation assay on
paired FF and FFPE tissue from 10 lymphoma samples
and 10 lymph node hyperplasia samples [23]. They
found good correlation of DML between FF and FFPE
in different groups, although the number of loci was
small. Even though Killian identified lymphoma-specific
DML in comparison with hyperplasia samples, the
lymphoma and hyperplasia samples were not paired
from the same patient (unlike our study, in which
tumor and adjacent normal samples were paired).
To our knowledge, our study is unique in addressing
this issue at a genome-wide level using a large number
of samples and a well-designed experiment to validate
tumor-specific DML data derived from paired FFPE
DNA (tumor and normal) against DML data from
paired corresponding FF tissue DNA. The discrepancy
between FFPE and FF samples in our study may reflect:
(a) the incompatibility of the Infinium chemistry, includ-
ing the WGA component; and/or (b) DNA damage
induced by tissue fixation, which may lead to misidenti-
fication or miscalculation of b values. Fixation-induced
changes in methylation status may be ruled out, since
previous authors’ GoldenGate data and other low-plex
methylation data did not find significant differences
between FFPE samples and corresponding FF samples.
Conclusions
In conclusion, ligation-based repair of FFPE DNA may
allow the Infinium whole-genome methylation assay to
Figure 10 Infinium genotyping data from paired FF and ligated FFPE samples of CRC and adjacent normal tissue. These samples are an
independent set from those used for Infinium methylation. (A) The genotype call rates for ligated FFPE samples were significantly lower and
more variable than those of the FF replicates. (B) Representative logR ratio (upper pane) and B-allele frequency (lower pane) from chromosome
1 from the FF sample from an individual patient (C_28) (C) Corresponding data for the FFPE replicate of the same patient in panel B, who had
the higher call rate in FFPE sample. (D) Representative logR ratio and B-allele frequency plots from chromosome 18 from the FF sample from a
second patient (C_34). (E) Corresponding data for the FFPE replicate from the same patient C_34 in panel D, who had lower call rates in FFPE
sample.
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detect a reasonable number of loci, although much
fewer than in unmodified FF DNA. Infinium methyla-
tion data from ligated FFPE DNA may also differentiate
tumor and normal samples, but the DML sets derived
from FFPE and FF samples are very discordant and may
not provide the same biological information. Therefore,
tumor-specific DML identified in FFPE tissue with this
method should be interpreted with great caution.
Additional material
Additional file 1: Figure S1. Distribution of signal intensities for loci in
FF tissue (red) and FFPE tissue (blue).
Additional file 2: Figure S2. Infinium methylation data from technical
replicates.
Additional file 3: Figure S3. Differentially methylated loci detected by
three statistical analyses.
Additional file 4: Table S1. The top 20 DML derived from multi-way
ANOVA of FF and FFPE DNA.
Additional file 5: Figure S4. Correlation of p-values of DML detected in
FF and FFPE tissue using ANOVA. Log10-transformed p-values from FF
samples are shown on the x-axis, and log10-transformed p-values from
FFPE samples are shown on the y-axis.
Additional file 6: Table S2. Correlation of b values from FF and FFPE
samples for all the methylation probes for selected five genes.
Acknowledgements
This work was supported by the National Institutes of Health grants U01
CA122171, P30 CA 014599, P42ES010349, R01CA102484, and R01CA107431.
Author details
1Department of Health Studies, The University of Chicago, Chicago, IL 60637,
USA.2Department of Human Genetics, The University of Chicago, Chicago, IL
60637, USA.3Department of Medicine, The University of Chicago, Chicago, IL
60637, USA.4Department of Comprehensive Cancer Center, The University of
Chicago, Chicago, IL 60637, USA.5University of Chicago Research Office in
Bangladesh, Dhaka, Bangladesh.6Department of Pathology, Bangabandhu
Sheikh Mujib Medical University (BSMMU), Dhaka 1000, Bangladesh.
Authors’ contributions
FJ designed and carried out the genome-wide methylation assay and wrote
the manuscript, RR drafted the manuscript, SR processed the tissue samples
and carried out the methylation and validation assay, MR and RP collected
the tissue samples and did the histopathology, MRZ helped in sample
collection and transportation of the samples to USA, RPB and CD helped in
methylation microarray, MK organized and supervised the tissue collection
and was responsible for histopathology, HA conceived and helped in study
design, manuscript preparation, supported and coordinated the study, MGK
conceived and designed the study, performed data analysis and wrote the
manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 7 November 2011 Accepted: 22 February 2012
Published: 22 February 2012
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doi:10.1186/1756-0500-5-117
Cite this article as: Jasmine et al.: Interpretation of genome-wide
infinium methylation data from ligated DNA in formalin-fixed, paraffin-
embedded paired tumor and normal tissue. BMC Research Notes 2012
5:117.
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