Analytical sensitivity and stability of DNA methylation testing in stool samples for colorectal cancer detection

• Published in: Cellular oncology (Dordrecht) Vol. 35; no. 4; pp. 309 - 315
• Main Authors: Bosch, Linda J. W., Mongera, Sandra, sive Droste, Jochim S. Terhaar, Oort, Frank A., van Turenhout, Sietze T., Penning, Maarten T., Louwagie, Joost, Mulder, Chris J. J., van Engeland, Manon, Carvalho, Beatriz, Meijer, Gerrit A.
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.08.2012
• Subjects: Base Sequence; beta-Globins - genetics; Biomedical and Life Sciences; Biomedicine; Cancer Research; Cell Cycle Proteins - genetics; Colorectal Neoplasms - diagnosis; Colorectal Neoplasms - genetics; DNA Methylation; DNA Mutational Analysis; DNA, Neoplasm - genetics; Feasibility Studies; Feces - chemistry; Genetic Testing - methods; HCT116 Cells; Humans; Mutation; Neoplasm Proteins - genetics; Oncology; Original Paper; Pathology; Poly-ADP-Ribose Binding Proteins; Polymerase Chain Reaction; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins p21(ras); ras Proteins - genetics; Reproducibility of Results; Sensitivity and Specificity; Temperature; Time Factors; Ubiquitin-Protein Ligases; FIT; Screening; Stool; Analytical sensitivity; Stability; CRC; DNA methylation; iFOBT
• ISSN: 2211-3428; 2211-3436; 2211-3436
• DOI: 10.1007/s13402-012-0092-6

Background Stool-based molecular tests hold large potential for improving colorectal cancer screening. Here, we investigated the analytical sensitivity of a DNA methylation assay on partial stool samples, and estimated the DNA degradation in stool over time. In addition, we explored the detection of DNA methylation in fecal immunochemical test (FIT) fluid. Materials and Methods Partial stool samples of colonoscopy-negative individuals were homogenized with stool homogenization buffer, spiked with different numbers of HCT116 colon cancer cells and kept at room temperature for 0, 24, 48, 72 and 144 h before DNA isolation. Analytical sensitivity was determined by the lowest number of cells that yielded positive test results by DNA methylation or mutation analysis. DNA methylation in FIT fluid was measured in 11 CRC patients and 20 control subjects. Results The analytical sensitivity for detecting DNA methylation was 3000 cells per gram stool, compared to 60000 cells per gram stool for detection of DNA mutations in the same stool samples. No degradation up to 72 h was noted when a conservation buffer was used. DNA methylation was detected in 4/11 CRC FIT samples and in none of the 20 control FIT samples. Conclusions Methylation based stool DNA testing showed a high analytical sensitivity for tumor DNA in partial stool samples, which was hardly influenced by DNA degradation over time, provided an adequate buffer was used. The feasibility of detecting DNA methylation in FIT fluid demonstrates the opportunity to combine testing for occult blood with DNA methylation in the same collection device.