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

Author(s): Jasmine F, Rahaman R, Roy S, Raza M, Paul R, Rakibuz-Zaman M, Paul-Brutus R, Dodsworth C, Kamal M, Ahsan H, Kibriya MG

Publication: BMC Res Notes, 2012, Vol. 5, Page 117

PubMed ID: 22357164 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to assess the accuracy of whole genome methylation data generated with tumor and normal adjacent formalin-fixed paraffin-embedded (FFPE) colon specimens based upon the results obtained with case-matched frozen controls.  DNA isolated from FFPE specimens was subjected to a modified ligation-based repair protocol prior to analysis with an Illumina Infinium Methylation 27K Chip.  Tumor and normal adjacent colon specimens were procured from 12 patients. 

   Summary of Findings:

   The number of loci detected by Illumina's Infinium Methylation 27K chip was significantly lower for FFPE specimens compare to case-matched frozen specimens for both normal adjacent (27,102 vs. 27,541; p=0.002) and tumor (27,239 vs. 27,530; p=0.032) specimens. The number of detected loci did not differ between tumor and normal adjacent specimens for either FFPE or frozen specimens.

   Principal component analysis revealed that samples clustered first by preservation method (FFPE, frozen) and then by tumor status (cancerous, normal adjacent), and the separation between tumor and normal adjacent specimens was less defined in FFPE than frozen specimens.  Distribution patterns for methylation levels were similar between FFPE and frozen, with the majority of loci detected as hypomethylated, although clustering was still evident. Patient-specific scatter plots revealed that methlyation status was more weakly correlated between FFPE and frozen specimens than expected for case-matched specimens (r²=0.78 for normal adjacent, r²=0.73 for a tumor specimen from a representative patient). Notably, stronger correlations were observed when samples from multiple patients were plotted together (r²=0.89 for normal adjacent, r²=0.93 for tumor specimens).  Only 10% of the 26,486 individual methylation loci detected displayed a strong correlation (r≥0.6) in β-values between case-matched FFPE and frozen specimens.  When the top 50 tumor-specific DML were compared between FFPE and frozen specimens, only 7 loci representing 6 genes (EYA4, TFPI2, GATA4, SPG20, WT1, SORC53) were common to both FFPE and frozen specimens. Despite the lack of concordance, samples from tumor and normal adjacent FFPE specimens were effectively separated when examined by unsupervised hierarchial clustering.  

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	DNA microarray
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen

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