Validation of the MethylationEPIC BeadChip for fresh-frozen and formalin-fixed paraffin-embedded tumours
© The Author(s). 2017
Received: 7 December 2016
Accepted: 24 March 2017
Published: 4 April 2017
DNA methylation is the most studied epigenetic modification due to its role in regulating gene expression, and its involvement in the pathogenesis of cancer and several diseases upon aberrations in methylation. The method of choice to evaluate genome-wide methylation has been the Illumina HumanMethylation450 BeadChip (450K), but it was recently replaced with the MethylationEPIC BeadChip (EPIC). We therefore sought to validate the EPIC array in comparison to the 450K array for both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tumours. We also performed analysis on the EPIC array with paired FF and FFPE samples to adapt to a clinical setting where FFPE is routinely used. Further, we compared two restoration methods, REPLI-g and Infinium, for FFPE-derived DNA on the EPIC array.
The Pearson correlation of β values for common probes on the 450K and EPIC array was high for both FF (mean: 0.992) and FFPE (mean: 0.984) samples. The β values generated from the EPIC array for FFPE samples correlated well with the paired FF tumours, but varied between 0.901 and 0.987. We did note that sample pairs with lower correlation had less bimodal density distributions of β values and displayed higher noise in the copy number alteration plots (generated from the methylation array data) in the FFPE sample. Both REPLI-g and the Infinium restoration for FFPE samples performed well on the EPIC array and generated equivalent correlation scores to the paired FF sample.
KeywordsEpigenetics DNA methylation 450K EPIC Brain tumour Formalin-fixed paraffin-embedded tissue DNA restoration
DNA methylation is the most studied epigenetic mark, as it is crucial for cell development and regulation of gene expression with aberrations involved in the pathogenesis of several diseases including cancer . DNA methylation is the addition of a methyl group to the carbon-5 position of cytosine, which in mammals primarily takes place on cytosine-phosphate-guanine (CpG) dinucleotides. Several CpG sites clustering in close proximity are termed CpG islands, which are most frequently located in gene promoter regions inferring that unmethylated CpG islands allow for transcription whereas hypermethylated promoter regions repress gene expression . DNA methylation aberrations, e.g. in the form of hypermethylation of the promoter of a tumour-supressing gene, are common in cancer, including paediatric brain tumours, and methylation profiling is an increasingly valuable tool when it comes to tumour classification [3–5]. The Illumina HumanMethylation450 BeadChip (450K), covering 485,000 CpG sites, has been the most common method to assess genome-wide methylation , but this array has now been discontinued and replaced with the MethylationEPIC BeadChip (EPIC) which covers 850,000 CpG sites .
The use of DNA methylation profiling to classify and subgroup paediatric brain tumours is now moving into clinical diagnostics where the golden standard for histopathological assessments and archiving material is formalin-fixed paraffin-embedded tissue (FFPE). While FFPE is excellent for this purpose, it does induce problems with extraction of nucleotides as the formalin-fixation causes cross-linking and fragmentation of the DNA thus yielding degraded DNA of poor quality . This degradation renders the DNA unsuitable for whole genome amplification (WGA), a vital step in Illumina methylation arrays, thus preventing the use of methylation arrays on FFPE samples. However, two different methods for DNA restoration produced satisfying results for FFPE samples using the 450K arrays [9–11]. To ascertain the transferability to the EPIC arrays, we have compared paediatric brain tumour samples that were analysed with the 450K and the EPIC arrays; both fresh-frozen (FF) and FFPE samples, thereby reporting the first validation of FFPE samples between the platforms. The EPIC array has thus far been validated in comparison with the 450K platform only for cell lines and blood samples  and merely one paired sample (renal tumour) of FF and FFPE . We therefore also assessed the accuracy of the EPIC array by comparing nine paired FF and FFPE brain tumour samples and also performed the first comparison on the EPIC array of the two aforementioned protocols for DNA restoration; REPLI-g  and Infinium .
To investigate the distribution of CpG sites that had differing β values in the FF samples compared to the FFPE samples, we specifically analysed all sites with a difference in β value above 0.2 between FF and FFPE samples from the same tumour. For each CpG site, we counted how many of the nine sample pairs that had a β difference >0.2. Very few of the differing sites (268) were present in all nine tumour FF-FFPE pairs, suggesting that CpG sites were affected at random in the FFPE samples making it impossible to predict which sites that are affected when not using paired samples. These 268 differing sites present in all nine samples were spread evenly over the chromosomes.
Number of failed probes (p > 0.01) in samples
Comparison of REPLI-g and Infinium DNA restoration of FFPE samples analysed on the EPIC array
FF vs FFPE REPLI-g (r)
FF vs FFPE Infinium (r)
We have investigated the performance of the new EPIC array compared to the 450K array for brain tumour samples, as well as evaluated the concordance in β values with the EPIC platform between FF and FFPE samples, thus adapting to a clinical setting where FFPE is the standard preservation method of tissue. We also evaluated two different DNA restoration protocols for FFPE samples. To conclude, the reproducibility of 450K data using the EPIC array is good for both FF and FFPE brain tumour samples. The EPIC array, like its predecessor, generates β values from restored FFPE samples similar to those of the paired FF samples. We note, however, that the correlation between paired FF and FFPE samples varies, where noise in the FFPE CNA plot and higher density in the low-mid range (0.2-0.4) region can be an indication of bad quality in the FFPE sample. Further, both evaluated DNA restoration methods yield satisfying results from FFPE samples on the EPIC array with very similar mean correlation scores to the matched FF samples. We can therefore not see any clear benefit for choosing one method over the other as both are suitable for the purpose of restoring FFPE-derived DNA for analysis on the EPIC array.
Materials and methods
Tumour tissue was obtained from children who underwent brain tumour surgery at Sahlgrenska University Hospital 2013–2015 after signed informed consent from the parents. Part of the tumour tissue was preserved as FF in liquid nitrogen and part of it as FFPE. FFPE samples were processed according to standard procedures at the department of Pathology at Sahlgrenska University Hospital by fixation in 4% formaldehyde for 24 or 48 h (depending on the size of the tissue) prior to dehydration with increasing ethanol concentrations and xylene before paraffin infiltration and embedment.
DNA isolation and quality control
DNA from FF tumours was extracted with the DNeasy Blood & Tissue Kit (69504, Qiagen, Hilden, Germany) according to the manufacturer’s instructions with the addition of lysing the samples with the QIAGEN TissueLyser. FFPE samples (1–3 years old) were sectioned, and DNA was isolated using QIAamp DNA FFPE Tissue Kit (56404, Qiagen) according to manufacturer’s recommendations except de-waxing steps with Xylene was repeated twice, and the tissue was digested overnight. DNA concentration was determined with the Qubit® Fluorometer (Life technologies). The quality of the DNA extracted from FFPE samples was assessed in triplicates with the real-time PCR-based Illumina FFPE QC kit (WG-321-1001, Illumina, Inc., San Diego, CA) according to the provided protocol. FFPE samples with delta CT < 3 compared to the QC template (supplied in kit) was deemed eligible for restoration with REPLI-g or Infinium restoration kit.
DNA from FFPE samples was restored either with (1) REPLI-g prior to bisulfite conversion or (2) with the Infinium restoration kit after bisulfite modification (see below). 1 μg FFPE DNA was used for REPLI-g restoration as described previously . For four out of the nine tumours with paired FF and FFPE, we performed restoration with REPLI-g and Illumina to compare the restoration procedures.
500 ng FF DNA, 1 μg REPLI-g-restored FFPE DNA and 1 μg-unrestored FFPE DNA (for restoration with the Infinium kit after bisulfite conversion) was used for bisulfite conversion with the EZ DNA methylation kit (D5001, Zymo Research, Orange, CA) according to the manufacturer’s instructions using the alternative incubation conditions recommended for the Illumina Infinium methylation arrays. Successful conversion was verified by control PCR reactions with a primer set specific for bisulfite-converted DNA, and a primer set for unconverted DNA.
All of eluate 1 of the bisulfite-converted unrestored FFPE DNA (n = 4 samples) was used for restoration with the Infinium HD FFPE DNA Restore Kit (WG-321-1002, Illumina) using the Infinium HD FFPE Restore Protocol supplied by the manufacturer.
Genome-wide methylation arrays
Infinium HumanMethylation450 BeadChips and Infinium MethylationEPIC BeadChips (Illumina) were used for the determination of methylation levels of more than 450,000 and 850,000 CpG sites, respectively, as previously described . 4 μl of eluate 1 of the FF-, REPLI-g-restored and Infinium-restored bisulfite-converted DNA was used for the methylation arrays according to Illumina’s protocols. Unrestored FFPE DNA was not processed on the methylation arrays.
Raw methylation data was normalised using Noob-normalisation with the R minfi package [15–17]. CpG sites with detection p value >0.01 were regarded as failed and were assigned as missing. Also, 43,254 CpG sites with probes identified to be cross-hybridizing according to Pidsley et al.  were removed prior to analysis. For the comparisons between the 450K and EPIC arrays, we analysed probes present on both platforms. CNA analysis was performed using the conumee R package . As reference samples for the CNA analysis, we used EPIC array data from three samples of non-malignant tissue-associated fibroblasts (NAF), and five samples of infant blood from archival Guthrie cards available in the public GEO dataset: GSE86831.
Illumina HumanMethylation450 BeadChip
Copy number alteration
Non-malignant tissue associated fibroblasts
Whole genome amplification
We thank UCL Genomics for DNA methylation array processing.
This work was supported by The Swedish Cancer Society, the Swedish Children’s Cancer Society, the Swedish Research Council, the Swedish Society for Medical Research, the Wenner-Gren foundation and a FP7 Marie Curie career integration grant.
Availability of data and materials
Data sets are accessible in GEO under the following accession: GSE92580.
HC initiated the study. AW performed the experimental work. TK performed the data analyses and prepared figures and tables. HC, AW and TK wrote the manuscript with input from SB. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The consent for publication was obtained from the ethical approval.
Ethics approval and consent to participate
The study was approved by the regional ethics committee (Dnr 604–12) and was carried out in accordance with the relevant guidelines and regulations.
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