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Updated: Jun 13, 2026
Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer
Published on: September 18, 2020
Immunoguided laser assisted microdissection techniques for DNA methylation analysis of archival tissue specimens
Franziska C Eberle1, Jeffrey C Hanson, J Keith Killian
1Hematopathology Section, National Cancer Institute, National Institutes of Health, 8717 Grovemont Circle, Bethesda, MD 20892, USA.
Abstract:
Altered DNA methylation is a fundamental characteristic of carcinogenesis. The analysis of DNA methylation in tumor cells may help to better understand tumor pathogenesis and more importantly may be used as diagnostic tool with therapeutic consequences. To detect targets relevant in tumorigenesis, it is essential to separate neoplastic cells from nonneoplastic cells. An excellent method for isolating specific cells is laser-assisted microdissection (LAM). Target cell identification for immunoguided LAM (ILAM) requires immunohistochemistry (IHC). Yet, it is unclear whether IHC for ILAM influences DNA methylation. The goals of this study were to establish an optimized protocol for antigen retrieval and IHC of formalin-fixed paraffin-embedded (FFPE) specimens suitable for ILAM and to evaluate its effect on the DNA methylome using a high throughput array. Using ten archival FFPE specimens, we showed specific staining suitable for ILAM. Extracted DNA from microdissected cells of immunohistochemically or H&E-stained tissue sections showed identical DNA quality and a strong correlation (r = 0.94 to 0.98) for CpG target methylation of 1505 analyzed sites in a series of five paired samples. No differential methylation between H&E and IHC was detected in 1501 of 1505 CpG targets (99.7%; P < 0.05). These results demonstrate the validity and utility of the herein described protocol, which allows the application of ILAM for large-scale genomic and epigenetic analyses of archival tissue specimens.
Insights
Immunohistochemistry (IHC) for laser-assisted microdissection (LAM) does not alter DNA methylation in formalin-fixed paraffin-embedded (FFPE) tissues. This validated protocol enables large-scale genomic and epigenetic analysis of archival specimens.
Area of Science:
- Epigenetics
- Molecular Biology
- Cancer Research
Background:
- Altered DNA methylation is a hallmark of cancer, offering potential diagnostic and therapeutic targets.
- Accurate analysis requires separating tumor cells from non-tumor cells, often using laser-assisted microdissection (LAM).
- Immunohistochemistry (IHC) is used for cell identification in immunoguided LAM (ILAM), but its impact on DNA methylation is unknown.
Purpose of the Study:
- To optimize antigen retrieval and IHC protocols for FFPE specimens compatible with ILAM.
- To evaluate the effect of IHC staining on the DNA methylome.
Main Methods:
- Developed and optimized an IHC protocol for FFPE tissue sections.
- Performed immunoguided LAM (ILAM) on stained sections.
- Extracted DNA from microdissected cells.
- Analyzed DNA methylation using a high-throughput array on paired H&E and IHC samples.
Main Results:
- Achieved specific staining suitable for ILAM.
- DNA extracted from microdissected cells showed identical quality and high correlation (r = 0.94–0.98) in CpG methylation between H&E and IHC samples.
- No significant differential methylation was detected in 99.7% of analyzed CpG targets (1501/1505).
Conclusions:
- The established IHC protocol is valid for ILAM.
- IHC staining does not significantly impact DNA methylation profiles.
- This method facilitates large-scale genomic and epigenetic analyses of archival FFPE tissues using ILAM.
