Early blood-based detection of colorectal cancer by methylation-specific droplet digital PCR - A clinical biomarker discovery and validation study

Sarah Østrup Jensen ¹ Nadia Øgaard ¹ Mai-Britt Worm Ørntoft ¹ Jesper Bertram Bramsen ¹ Mads Heilskov Rasmussen ¹ Helle Kristensen ² Peter Mouritzen ² Mogens Rørbæk Madsen ³ Anders Husted Madsen ³ Kåre Gotschalck Sunesen ⁴ Lene Hjerrild Iversen ⁵ Søren Laurberg ⁵ Ib Jarle Christensen ⁶ Hans Jørgen Nielsen ⁶ Claus Lindbjerg Andersen ¹

¹Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark
²Exiqon, Qiagen, Vedbæk, Denmark
³Department of Surgery, Herning Regional Hospital, Herning, Denmark
⁴Department of Surgery, Randers Regional Hospital, Randers, Denmark
⁵Department of Surgery, Aarhus University Hospital, Aarhus, Denmark
⁶Department of Surgical Gastroenterology, Hvidovre Hospital, Hvidovre, Denmark

PURPOSE:

Early detection plays an essential role to reduce colorectal cancer mortality. 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 tumor-specific DNA methylation markers in circulating cell-free DNA.

METHODS:

The markers were identified in a thorough multi-step biomarker discovery process based on DNA methylation profiles of more than 5,000 tumors and blood cell populations. Using methylation-specific droplet digital PCR, the TriMeth test was 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 TriMeth was validated in an independent cohort comprising 143 CRC patients and 91 controls.

RESULTS:

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 II, 89% stage III (49/55), and 88% stage IV (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 underlines the potential utility of DNA methylation-based detection of circulating tumor DNA in the clinical management of colorectal cancer.