Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis
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Updated: May 3, 2026
Immunostaining for DNA Modifications: Computational Analysis of Confocal Images
Published on: September 7, 2017
F J Hernandez-Blazquez1, M Habib, J M Dumollard
1Faculdade de Zootecnia e Engenharia de Alimentos, Universidad de São Paulo, Pirassununga (SP), Brazil.
Background:
Global hypomethylation of DNA is frequently observed in human tumours. This alteration is detected in early adenomas in colorectal tumorigenesis. Information is currently acquired after extraction of DNA from tissues, digestion with nucleases, and analysis by reverse phase chromatography, or treatment with restriction enzymes followed by gel electrophoresis analysis and Southern hybridisation with radiolabelled probes.
Aims:
The purpose of our work was to evaluate the global methylation status of DNA in malignant lesions without loosing the histopathological features of the samples.
Patients:
The investigation was performed on paired normal-tumour tissues from 13 patients undergoing surgical resection of colorectal adenocarcinomas.
Methods:
Antibodies raised against 5-methylcytidine can be used to label methyl rich regions in interphase nuclei. This technique was adapted to the study of paraffin embedded tissues and an immunohistochemical method was developed to assess the global methylation status of individual nuclei while preserving cell morphology and tissue architecture. Computer assisted quantification of the staining intensity was performed on malignant and normal zones of human colon tissues to test the correlation between the immunolabelling signal and the respective histological patterns observed.
Results:
Qualitative and quantitative differences were observed and measured between the normal and malignant part of each sample. Morphologically altered nuclei displayed densely labelled spots within faintly labelled areas whereas normal nuclei were darker and uniformly stained. Image analysis allowed calculation of the average integrated optical density of the nuclei in both types of tissues, demonstrating a constant and significantly lower intensity for the former type of cells.
Insights
Global DNA hypomethylation is a hallmark of cancer. This study developed an immunohistochemical method to assess DNA methylation in colorectal cancer tissues, revealing significant differences between normal and malignant cells.
Area of Science:
- Oncology
- Epigenetics
- Molecular Biology
Background:
- Global DNA hypomethylation is a common feature in human tumors, including early colorectal adenomas.
- Current methods for DNA methylation analysis are destructive, losing histopathological context.
Purpose of the Study:
- To evaluate the global DNA methylation status in malignant colorectal lesions.
- To preserve histopathological features during methylation analysis.
Main Methods:
- Developed an immunohistochemical technique using anti-5-methylcytidine antibodies on paraffin-embedded tissues.
- Applied the method to paired normal and colorectal adenocarcinoma tissues from 13 patients.
- Utilized computer-assisted image analysis to quantify staining intensity in relation to histological patterns.
Main Results:
- Observed distinct qualitative and quantitative differences in DNA methylation between normal and malignant colorectal tissues.
- Malignant nuclei showed altered staining patterns with densely labeled spots in faint areas, unlike uniformly stained normal nuclei.
- Image analysis confirmed significantly lower average integrated optical density in malignant nuclei compared to normal ones.
Conclusions:
- The developed immunohistochemical method accurately assesses global DNA methylation status while preserving tissue morphology.
- This technique allows for the correlation of DNA methylation changes with histopathological features in colorectal cancer.
