Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays.
Author(s): de Ruijter TC, de Hoon JP, Slaats J, de Vries B, Janssen MJ, van Wezel T, Aarts MJ, van Engeland M, Tjan-Heijnen VC, Van Neste L, Veeck J
Publication: Lab Invest, 2015, Vol. 95, Page 833-42
PubMed ID: 25867767 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if DNA restoration improves concordance in genotyping and methylation status between matched formalin-fixed and paraffin-embedded (FFPE) and snap-frozen specimens. The potential impact of FFPE block storage on reproducibility was also investigated.
Conclusion of Paper
Compared to frozen specimens, FFPE specimens had lower signal intensity, lower probe detection rates and lower concordance in genotype and methylation status between technical replicates, but restoration of FFPE specimens partially attenuated these effects. Further, the discordance in genotyping and methylation status was lower between matched frozen and restored FFPE specimens than between frozen and unrestored FFPE specimens. When the threshold for methylation probe discordance was increased to a β-value of >0.2, then discordance was <1% between technical replicates of frozen or restored FFPE specimens, and 5.7% between matched restored FFPE and frozen specimens, but was 10.9% between technical replicates of unrestored FFPE specimens, and 24.69% between matched unrestored FFPE and frozen specimens. Specimen age did not affect assay reproducibility.
Studies
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Study Purpose
The purpose of this study was to determine if DNA restoration can improve the concordance in genotyping and methylation status between matched FFPE and snap-frozen specimens, and to determine if reproducibility is influenced by FFPE block storage duration. DNA was extracted from eight 10 µm sections of 9 FFPE normal colonic mucosa specimens (stored for 3-25 years) and 5 matched snap-frozen specimen using the NucleoSpin FFPE Tissue and NucleoSpin tissue kits, respectively. DNA was extracted from eight 10 µm sections of 11 FFPE breast cancer specimens using the Maxwell FFPE CSC kit. Extracted DNA was restored using the Illumina HD FFPE Restoration Kit and analyzed using the Infinium HumanMethylation450 BeadChip assay (HM-450K). The approach was validated with an additional 12 FFPE normal colon specimens (archived 6-18 years) and 12 FFPE breast tumors archived 7-10 years.
Summary of Findings:
The mean fluorescent intensity of the probe signals and probe detection rates were highest for frozen specimens (14187 and 99.98%, respectively) followed by restored FFPE specimens (9434 and 98.37%, respectively) and unrestored FFPE specimens (1000-6000 and 82.31%, respectively). The Spearman’s correlation coefficient for genotyping between technical replicates was greater for snap-frozen specimens (ρ=0.9978) than for FFPE specimens (ρ=0.8960), but comparable to restored FFPE specimens (ρ=0.9891). Concordance in genotyping results between snap-frozen and restored FFPE specimens for 66 SNPs was ρ=0.986, while concordance between matched unrestored FFPE and frozen specimens was only ρ=0.788. While the mean β-values were lower (indicating a loss of methylation) for unrestored FFPE specimens compared to frozen specimens (p=0.02), differences in β-values between restored FFPE specimens and frozen specimens were not significant. When the threshold for methylation probe discordance was set to a β-value of 0.1, discordance between technical replicates of frozen, unrestored FFPE and restored FFPE specimens was 0.6%, 30.8% and 5.7%, respectively; but, when the threshold for discordance was increased to a β-value of 0.2, then discordance between technical replicates was <1%, 10.9% and <1%, respectively. Correlation coefficients for methylation levels were higher between frozen and restored FFPE specimens than between frozen and unrestored FFPE specimens (ρ=0.9590 versus ρ=0.8051), while discordance in methylation status (β value difference >0.2) between frozen and restored or unrestored FPPE specimens was 5.7% and 24.69%, respectively. Importantly, methylation results for 4 of 5 targets were also validated in additional restored FFPE specimens using methylation specific PCR (MSP); as the fifth target (CKLF-like MARVEL transmembrane domain-containing protein 2, CMTM2) produced discrepant results for colon specimens. Specimen age did not affect assay reproducibility, although only those specimens stored 3-10 years were analyzed.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Bisulfite conversion assay DNA PCR DNA DNA microarray Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Snap frozen
Storage Storage duration 3-10 years
PCR Specific Targeted nucleic acid GFI1
MX2
VWCE
NEU1
CMTM2
DNA microarray Specific Template modification Restored
Unrestored