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

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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.

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.

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Abbreviations

CHFR :

Checkpoint with forkhead and ring finger domains

CRC:

Colorectal cancer

FIT:

Fecal immunochemical test

KRAS :

Kirsten rat sarcoma viral oncogene homolog

nMSP / QMSP:

Nested / quantitative methylation specific PCR

PHACTR3:

Phosphatase and actin regulator 3

ACTB:

Beta actin

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Financial support

This study was supported by SenterNovem IS052034

Conflict of interest

MT Pennings and J Louwagie were employees of OncoMethylome Sciences and are holding stock options. GA Meijer is a consultant to MDxHealth (former OncoMethylome Sciences) and received research funding.

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Authors and Affiliations

  1. Department of Pathology, VU University Medical Center, PO Box 7057, 1007 MB, Amsterdam, The Netherlands

    Linda J. W. Bosch, Sandra Mongera, Beatriz Carvalho & Gerrit A. Meijer

  2. Department of Gastroenterology, VU University Medical Center, Amsterdam, The Netherlands

    Jochim S. Terhaar sive Droste, Frank A. Oort, Sietze T. van Turenhout & Chris J. J. Mulder

  3. OncoMethylome Sciences, BV, Amsterdam, The Netherlands

    Maarten T. Penning

  4. OncoMethylome Sciences, SA, Liège, Belgium

    Joost Louwagie

  5. Department of Pathology, GROW - School for Oncology and Developmental Biology and Maastricht University Medical Center, Maastricht, The Netherlands

    Manon van Engeland

Authors
  1. Linda J. W. Bosch
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  2. Sandra Mongera
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  10. Beatriz Carvalho
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  11. Gerrit A. Meijer
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Corresponding author

Correspondence to Gerrit A. Meijer.

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Bosch, L.J.W., Mongera, S., sive Droste, J.S.T. et al. Analytical sensitivity and stability of DNA methylation testing in stool samples for colorectal cancer detection. Cell Oncol. 35, 309–315 (2012). https://doi.org/10.1007/s13402-012-0092-6

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