One of the important shortcomings in the published CRC biomarker studies is the reliance on a candidate gene approach for marker discovery. This approach is often based on a nonsystematic selection of candidate marker genes, which are tested in healthy and cancerous tissues and then validated in a patient population 
. Although some of these studies have resulted in promising biomarkers for early CRC detection, the lack of a thorough biomarker discovery strategy raises the question whether superior markers may have been overlooked. With the more advanced technologies currently available, it is possible to obtain genome-scale DNA methylation data that can be useful for biomarker discovery. We performed a genome-scale multistep marker discovery to identify CRC biomarkers using the HM27 and HM450 BeadChip platform; the latter evaluates the DNA methylation status of over 482,000 CpG loci and covers 96% of all UCSC CpG islands. We recently conducted a similar marker-pipeline strategy for ovarian cancer and identified the new sensitive recurrence biomarker IFFO1-M 
. For the present study we improved our discovery strategy by using DNA methylation data from 4,201 cancer samples of different origins to optimize CRC specificity. Our findings show that this discovery strategy works successfully for CRC, resulting in two new biomarkers: THBD-M and C9orf50-M. With AUCs of 0.97 and 1.0 respectively on the Infinium assay, these two markers have an excellent ability to distinguish between CRC tumors and matched normal colon tissue. Although DNA methylation of these genes has not yet been reported in association with CRC early detection, a recent study showed that aberrant THBD
DNA methylation was linked to gastric cancer carcinogenesis 
, which is consistent with the slightly higher DNA methylation levels observed for this marker in gastric cancer samples compared to other types of cancer (). Nevertheless, we found significantly higher levels of THBD
DNA methylation in CRC than in gastric cancer (p<0.001). In this study we did not explore the biological relevance of methylated THBD
in CRC carcinogenesis. The purpose of this study was to identify markers with high sensitivity and specificity, regardless of their function. Indeed, we anticipate that many cancer-associated epigenetic changes may represent passenger events, rather than drivers of oncogenesis.
The application of Digital PCR to multiplexed MethyLight assays allowed for efficient use of valuable samples by simultaneously analyzing more than one marker without loss of sensitivity. This technology allows for the detection of single methylated DNA molecules against a large background of unmethylated molecules, and provides a quantitative PCR test result 
Circulating free cancer DNA (cfDNA) has the potential to be tumor-specific and has a relatively short half-life making it suitable as biomarker 
. Although our two markers were consistently methylated in almost all CRC tumors, demonstrated excellent tissue-based discrimination between tumor and normal mucosa and tested positive in 25 clinical CRC samples using MethyLight, we were unable to detect DNA methylated at these loci in 1 ml samples of peripheral serum or plasma for some CRC cases. It is conceivable that the use of larger analyte volumes would increase sensitivity 
, but some tumors may not shed substantial amounts of tumor-derived DNA into the bloodstream. It is thought that tumor DNA enters the bloodstream by secretion or as a result of apoptosis or necrosis of cancer cells from the primary tumor or metastatic deposits 
. While cancer patients tend to have higher cfDNA levels than healthy subjects, concentrations of cfDNA in peripheral blood may vary significantly between individuals 
. In a prior study on Digital Methylight PCR we showed that DNA methylation marker levels in plasma of breast cancer patients are not directly related to the total yield of cfDNA as detected by an ALU reaction. For this reason we did not measure the amount of recovered cfDNA before performing Digital Methylight and chose to compare equal volumes of analyte that were processed in a standardized protocol 
One of the technical factors that could influence diagnostic performance of a biomarker is test volume. For example, the SEPT9
assay utilizes 4–5 ml of plasma 
. A few studies, however, have demonstrated that SEPT9
is also methylated in other types of cancer such as in lung adenocarcinoma 
, breast cancer 
and head and neck squamous cell carcinoma 
. We showed that THBD
harbor low levels of DNA methylation in 15 types of cancer other than CRC, including most high-incidence cancers. We anticipate that further assay optimization should produce substantially improved marker performance for both THBD-M and C9orf50-M.
While in this study, the use of serum resulted in a slightly higher test performance of THBD-M and the multiplex compared to plasma, this difference was of borderline significance. Although it has been reported that serum contains more cfDNA than plasma, no large-scale studies have been published comparing serum and plasma as test medium for blood-based detection of malignant diseases. Hence, it remains unclear whether serum or plasma is the optimal test specimen 
THBD-M outperformed C9orf50-M, and combining the two markers in a multiplexed assay did not increase test sensitivity. With a detection threshold of zero molecules per 1 ml plasma, THBD-M was able to detect 71% of all CRCs at a specificity of 80%. Interestingly, for stage I/II the detection rate in CRC was 74% with this marker. THBD-M had a higher sensitivity for the detection of colon cancers (77% for all stages) than rectal tumors (53% for all stages) in plasma. This difference was marginally significant (p
0.07). Early stage colon cancers were also detected by this marker at a relatively high percentage, 75% for stage I, and 77% for stage II. It is known that a subset of right-sided colon tumors exhibits high frequency of DNA hypermethylation at multiple promoter CpG islands, which is designated as CIMP 
. In addition it has been described that CIMP-high frequency increases gradually from the rectum to the right-sided colon 
. We did not observe significant differences in DNA methylation of THBD-M or C9orf50-M between CIMP and non-CIMP colorectal tumors. Both markers showed high levels of DNA methylation in all colorectal cancers, regardless of their CIMP status.
The fact that this diagnostic test detected a considerable fraction of mostly curable CRCs, with 5-year survival rates of 72%–93% 
, seems promising. With an AUC of 0.80 in plasma and 0.82 in serum, THBD-M compares favorably to or outperforms other published blood-based DNA methylation biomarkers 
Currently, no blood-based markers have yet been approved by the FDA for the use of early detection of CRC. Serum CEA is the only blood-based biomarker that is in use for CRC detection, but it lacks the sensitivity for primary CRC detection. Serum CEA measurement is used mainly as a follow-up tool after initial treatment, and yields a sensitivity of approximately 72% for the detection of liver metastasis and 60% for local recurrence with specificities of 91% and 86% respectively 
. In the present study, serum CEA detected only 33% of the primary CRCs.
In conclusion, we identified two novel blood-based DNA methylation markers for early detection of CRC though a systematic genome-scale marker discovery and verification study. Of these two markers, THBD-M had a promising performance in clinical samples justifying its further optimization and clinical testing.