title: Analytical sensitivity and stability of DNA methylation testing in stool samples for colorectal cancer detection.
abstract: 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.
  
  
  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.
authors: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands.
authors: Linda J W
authors: LJ
authors: Bosch
authors: Sandra
authors: S
authors: Mongera
authors: Jochim S
authors: JS
authors: Terhaar Sive Droste
authors: Frank A
authors: FA
authors: Oort
authors: Sietze T
authors: ST
authors: van Turenhout
authors: Maarten T
authors: MT
authors: Penning
authors: Joost
authors: J
authors: Louwagie
authors: Chris J J
authors: CJ
authors: Mulder
authors: Manon
authors: M
authors: van Engeland
authors: Beatriz
authors: B
authors: Carvalho
authors: Gerrit A
authors: GA
authors: Meijer
mesh_terms: D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics; D005240:Feasibility Studies; D005243:Feces / Q000737:chemistry*; D005820:Genetic Testing / Q000379:methods; D045325:HCT116 Cells; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D000075223:Poly-ADP-Ribose Binding Proteins; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases; D055544:beta-Globins / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011518:Proto-Oncogene Proteins; D055544:beta-Globins; C412002:CHFR protein, human; D044767:Ubiquitin-Protein Ligases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins