title: Highly sensitive faecal DNA testing of TWIST1 methylation in combination with faecal immunochemical test for haemoglobin is a promising marker for detection of colorectal neoplasia.
abstract: Background As TWIST1 methylation is specific to colorectal neoplasia, detection of TWIST1 methylation from faeces samples might be useful for colorectal neoplasia screening. However, because the content of human DNA in faeces is very small, it is very difficult to detect TWIST1 methylation by conventional bisulphite-based methylation assays. Therefore, we developed a new methylation assay without bisulphite treatment, the combined restriction digital PCR assay, and evaluated its sensitivity and specificity in combination with and without the faecal immunochemical test for haemoglobin for colorectal neoplasia detection from faeces samples. Methods For the combined restriction digital PCR assay, DNA was treated with three methylation-sensitive restriction enzymes and an exonuclease, followed by measurement of TWIST1 methylation level by droplet digital PCR. Faecal DNA testing and faecal immunochemical test for haemoglobin were performed on 109 patients with colorectal neoplasia and 10 control individuals. Results Basic performance testing showed that the combined restriction digital PCR assay enabled detection of 0.14% of the TWIST1 methylation level for the lymphocyte DNA. The combined restriction digital PCR assay from faeces samples had a sensitivity of 22.2% (95% confidence interval, 2.8-60.0%) for non-advanced adenoma, 47.1% (95% confidence interval, 23.0-72.2%) for advanced adenoma, and 33.7% (95% confidence interval, 23.7-45.0%) for colorectal cancer, and a specificity of 100.0%. Combination of faecal immunochemical test for haemoglobin and faecal combined restriction digital PCR assay increased sensitivity to 82.4% (95% confidence interval, 56.6-96.2%) for the detection of advanced adenoma. Conclusions We developed the combined restriction digital PCR assay, a possible highly sensitive methylation assay. Combination of faecal combined restriction digital PCR assay with faecal immunochemical test for haemoglobin may provide an alternative screening strategy for colorectal neoplasia, especially for potentially precancerous lesions.
authors.affiliation: 1 Department of Oncology and Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Suehiro
authors.affiliation: 1 Department of Oncology and Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Yibo
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: 2 Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Hashimoto
authors.affiliation: 2 Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Takami
authors.affiliation: 3 Department of Gastroenterology, Sentohiru Hospital, Ube, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Higaki
authors.affiliation: 4 Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Yoshitaro
authors.initials: Y
authors.lastname: Shindo
authors.affiliation: 4 Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Suzuki
authors.affiliation: 5 Department of Translational Research and Developmental Therapeutics against Cancer, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Shoichi
authors.initials: S
authors.lastname: Hazama
authors.affiliation: 4 Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oka
authors.affiliation: 4 Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nagano
authors.affiliation: 2 Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Sakaida
authors.affiliation: 1 Department of Oncology and Laboratory Medicine, Yamaguchi University Graduate School of Medicine, Ube, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamasaki
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000378:metabolism*; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000175:diagnosis*; D004247:DNA / Q000235:genetics*; D005243:Feces; D005260:Female; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009687:Nuclear Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D051879:Twist-Related Protein 1 / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D006454:Hemoglobins; D009687:Nuclear Proteins; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1; D004247:DNA
keywords: Advanced adenoma; TWIST1; colorectal cancer; digital PCR; faecal DNA test; faecal immunochemical test; methylation