Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study

Sarah Østrup Jensen, Nadia Øgaard, Mai-Britt Worm Ørntoft, Mads Heilskov Rasmussen, Jesper Bertram Bramsen, Helle Kristensen, Peter Mouritzen, Mogens Rørbæk Madsen, Anders Husted Madsen, Kåre Gotschalck Sunesen, Lene Hjerrild Iversen, Søren Laurberg, Ib Jarle Christensen, Hans Jørgen Nielsen, Claus Lindbjerg Andersen, Sarah Østrup Jensen, Nadia Øgaard, Mai-Britt Worm Ørntoft, Mads Heilskov Rasmussen, Jesper Bertram Bramsen, Helle Kristensen, Peter Mouritzen, Mogens Rørbæk Madsen, Anders Husted Madsen, Kåre Gotschalck Sunesen, Lene Hjerrild Iversen, Søren Laurberg, Ib Jarle Christensen, Hans Jørgen Nielsen, Claus Lindbjerg Andersen

Abstract

Background: Early detection plays an essential role to reduce colorectal cancer (CRC) mortality. While current screening methods suffer from poor compliance, liquid biopsy-based strategies for cancer detection is rapidly gaining promise. Here, we describe the development of TriMeth, a minimal-invasive blood-based test for detection of early-stage colorectal cancer. The test is based on assessment of three tumour-specific DNA methylation markers in circulating cell-free DNA.

Results: A thorough multi-step biomarker discovery study based on DNA methylation profiles of more than 5000 tumours and blood cell populations identified CRC-specific DNA methylation markers. The DNA methylation patterns of biomarker candidates were validated by bisulfite sequencing and methylation-specific droplet digital PCR in CRC tumour tissue and peripheral blood leucocytes. The three best performing markers were first applied to plasma from 113 primarily early-stage CRC patients and 87 age- and gender-matched colonoscopy-verified controls. Based on this, the test scoring algorithm was locked, and then TriMeth was validated in an independent cohort comprising 143 CRC patients and 91 controls. Three DNA methylation markers, C9orf50, KCNQ5, and CLIP4, were identified, each capable of discriminating plasma from colorectal cancer patients and healthy individuals (areas under the curve 0.86, 0.91, and 0.88). When combined in the TriMeth test, an average sensitivity of 85% (218/256) was observed (stage I: 80% (33/41), stage II: 85% (121/143), stage III: 89% (49/55), and stage IV: 88% (15/17)) at 99% (176/178) specificity in two independent plasma cohorts.

Conclusion: TriMeth enables detection of early-stage colorectal cancer with high sensitivity and specificity. The reported results underline the potential utility of DNA methylation-based detection of circulating tumour DNA in the clinical management of colorectal cancer.

Keywords: Cancer; Circulating tumour DNA; Colorectal cancer; DNA methylation; Early detection; Epigenetic biomarkers; Liquid biopsy.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Overview of study workflow. Infinium Human Methylation450K BeadChip® array data from > 4000 samples including CRC, PBL, normal colorectal mucosa, and other cancer types were used to identify CRC-specific DNA methylation biomarkers. The methylation pattern of candidate marker regions was confirmed by bisulfite Sanger sequencing of paired CRC tissue, normal colorectal mucosa, and PBLs. Methylation-specific ddPCR assays targeting candidate regions were designed and optimized, and clinical validation was performed by evaluating assays in CRC tumour tissue (n = 36) and PBL from blood donors (n = 27). The top three candidates were analysed in two independent cohorts consisting of plasma from CRC patients and controls. CRC colorectal cancer, PBLs peripheral blood leucocytes, ddPCR droplet digital PCR
Fig. 2
Fig. 2
Schematic representation of biomarker discovery and validation pipeline. Infinium HumanMethylation450K Beadchip® array data were used to evaluate the methylation status of CpG sites in CRC, PBL, normal mucosa, and other cancer types (left panel). We excluded CpG sites that were methylated in blood cells and CpG sites with low methylation in CRC. The remaining 6700 CpG sites were ranked according to CRC sensitivity and specificity against other common cancers. To confirm uniform methylation in genomic regions of candidate CpG sites, bisulfite Sanger sequencing was performed on paired samples of CRC, PBL, and normal colorectal mucosa. Twenty-nine of the top 50 CpG sites were located in regions compatible with successful methylation-specific ddPCR assay design. A total of 58 methylation-specific ddPCR assays were designed for the 29 CpG sites (markers) and tested in a sequence of validation steps (right panel). Assays were excluded if their performance was suboptimal in methylated and unmethylated control DNA, PBLs, and CRC tissue or in plasma from CRC patients. Three markers passed all selection criteria. CRC colorectal cancer, PBL peripheral blood leukocytes, ddPCR droplet digital PCR
Fig. 3
Fig. 3
DNA methylation of C9orf50, KCNQ5, and CLIP4. a DNA methylation levels (Infinium HumanMethylation450K BeadChip® array data) of the three markers C9orf50, KCNQ5, and CLIP4 in 571 individual CRC tumours, 556 PBL samples, and 4111 samples from other cancer types. Each of the three markers are hypermethylated (β-value > 0.35) in > 97% of CRC tumours. b Correlation of DNA methylation levels in CRC tissue of the C9orf50, KCNQ5, and CLIP4 markers. HNSCC head and neck squamous cell carcinoma, BCL B-Cell lymphoma, AML acute myeloid leukaemia
Fig. 4
Fig. 4
Detection of C9orf50, KCNQ5, and CLIP4 markers in plasma (test cohort). a Methylation-specific ddPCR was performed on 4500 copies of bisulfite-converted cfDNA to detect methylated C9orf50, KCNQ5, and CLIP4 in plasma samples from CRC patients and controls in the test cohort. The total number of methylated DNA copies (sum of the three markers) are recorded on the y-axis and CRC samples are arranged by UICC stage. b–d ROC curves (right) from test of C9orf50, KCNQ5, and CLIP4 individual markers in plasma. The red dots and dashed lines indicate the sensitivity and specificity when calling samples positive if they contained any methylated DNA. e Sensitivity and specificity by the TriMeth test in plasma. f UICC stage-stratified sensitivity of TriMeth in plasm. Error bars represent 95% CI. ddPCR droplet digital PCR, CRC colorectal cancer, UICC Union for International Cancer Control, ROC receiver operating characteristics
Fig. 5
Fig. 5
Independent validation of TriMeth in plasma. a Methylation-specific ddPCR was performed on cfDNA purified from plasma of CRC patients and controls (validation cohort) to detect methylated C9orf50, KCNQ5, and CLIP4. b Sensitivity and specificity of the TriMeth tes. c UICC stage-stratified sensitivity of TriMeth. Error bars represent 95% CI. ddPCR droplet digital PCR, CRC colorectal cancer, UICC Union for International Cancer Control

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