Why do results conflict regarding the prognostic value of the methylation status in colon cancers? The role of the preservation method.
Author(s): Tournier B, Chapusot C, Courcet E, Martin L, Lepage C, Faivre J, Piard F
Publication: BMC Cancer, 2012, Vol. 12, Page 12
PubMed ID: 22243995 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare methylation levels and bisulfite conversion assay efficacy between case-matched frozen and formalin-fixed, paraffin-embedded (FFPE) colon adenocarcinoma specimens. DNA extraction methods were also compared for FFPE specimens.
Conclusion of Paper
The percentage of specimens that generated interpretable results following bisulfite conversion was lower for FFPE specimens (55-67.5%) compared to case-matched frozen controls (87.2-97.4%), and was dependent upon the promoter investigated. In FFPE specimens, DNA extraction method also influenced the efficacy of bisulfite conversion of the LINE-1 promoter, as a higher percentage of interpretable results were obtained when a FFPE-specific extraction kit was used (75 vs. 55%), although values were still sub-par to frozen controls (95%). Although data was not shown, the authors note that the poor bisulfate conversion associated with extraction with a conventional kit led to artificially high levels of methylation compared to those observed among specimens extracted with the FFPE-specific kit. Methylation levels also differed between FFPE and frozen specimens, although no trends were observable among promoters.
Studies
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Study Purpose
The purpose of this study was to compare the efficacy and reliability of bisulfite conversion with pyrosequencing (an assay to quantify and detect DNA methylation) in FFPE specimens extracted using two different kits. A total of 40 surgically resected colon adenocarcinoma specimens were used. Case-matched specimens were extracted with a conventional magnetic-silica particle based kit (MagnaZorb) and the QIAamp DNA FFPE tissue kit (which includes a 1 h incubation at 90°C), and the percentage of interpretable results was recorded for a single promoter (LINE-1). Assays were run in duplicate for each sample.
Summary of Findings:
The efficacy of bisulfite conversion in FFPE specimens was affected by DNA extraction method, as the percentage of interpretable results was lower with the conventional magnetic-silica particle-based kit (55%; MagnaZorb kit by Bionobis) compared to the FFPE-specific kit (75%, QIAamp DNA FFPE tissue kit by Qiagen). Reliability was inferior among FFPE specimens extracted with a traditional kit when compared to the FFPE-specific extraction kit; 13/40 specimens extracted with the conventional kit displayed different pass/fail results between runs compared to 5/40 specimens extracted with the FFPE-specific kit. Although data was not shown, the authors note that the poor bisulfate conversion associated with extraction with a conventional kit led to artificially high levels of methylation compared to those observed among specimens extracted with the FFPE-specific kit.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA DNA sequencing DNA Bisulfite conversion assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAamp DNA FFPE Tissue kit (Qiagen)
MagaZorb kit
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Study Purpose
The purpose of this study was to compare promoter methylation levels for LINE-1, MLH1, and MGMT, as well as the efficacy of bisulfite conversion using pyrosequencing in case-matched frozen and FFPE colon adenocarcinoma specimens. A total of 40 surgically resected specimens were used, and assays were run in duplicate for each sample. DNA was extracted from frozen specimens with the MagZorb kit, and from FFPE specimens with the QIAamp DNA FFPE Tissue kit.
Summary of Findings:
The efficacy of bisulfite conversion and subsequent detection by pyrosequencing was adversely affected by formalin fixation and paraffin processing in comparison to matched frozen controls. The percentage of specimens that yielded interpretable results was lower among FFPE specimens than frozen specimen for all three promoters (LINE-1, 67.5 vs. 95%; MGMT, 55 vs. 87.2%; MLH, 57.5 vs. 97.4%). Further for MLH1, PCR amplification failed in 11 FFPE specimens while all 40 frozen specimens were amplifiable. Based upon the author-defined differential threshold of 6% (which exceeds inter-assay variability), significantly different methylation levels were observed among 27.6%, 15.4%, and 25.8% of case-matched FFPE/frozen specimens for LINE-1, MLH1, and MGMT, respectively. The magnitude and direction of the difference in methylation levels between matched FFPE and frozen specimens varied among specimens, with no observable trend among promoters. No specimens displayed significant differences in methylation levels for all three promoters; however, only 12.5% (5/40) specimens displayed both satisfactory controls for bisulfite conversion and no difference in methylation levels between FFPE and frozen specimens. Given the differences in assay efficacy and methlyation levels between FFPE and frozen specimens and the hetergenous efficacy results observed between markers for FFPE specimens, the authors postulate that for FFPE specimens the completeness of bisulfate conversion may differ from one cytosine to another, which may be an artifact of formalin-induced cross-linking.
Biospecimens
Preservative Types
- Formalin
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA DNA sequencing DNA Bisulfite conversion assay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen