J Mol Diagn. 2013 Jul; 15(4):498-507.

Genome-wide identification and validation of a novel methylation biomarker, SDC2, for blood-based detection of colorectal cancer.

Names of Authors: TaeJeong Oh*, Na-Young Kim*, Youngho Moon*, Myung Soon Kim*, Benjamin Douglass Hoehn*, Chan Hee Park, Tae Soo Kim, Nam Kyu Kim§, Hyun Cheol Chung†, ‡, ¶,Sungwhan An*

Affiliations of Authors

*Genomictree, Inc. 829 Tamnip-dong Yuseong Daejeon, 305-510, South Korea National Biochip Research Center, Yonsei University College of Medicine, 120-752, Seoul, South Korea Cancer Metastasis Research Center, Yonsei University College of Medicine, Seoul, 120-752, South Korea§Department of Surgery, Yonsei University College of Medicine, Seoul, 120-752, South KoreaDepartment of Internal Medicine, Yonsei University College of Medicine, Seoul, 120-752, South Korea

 

Abstract

Aberrant DNA methylation has shown promise as a biomarker for the early detection of cancer. To discover novel genes frequently methylated at an early stage in colorectal cancer (CRC), DNA microarray analysis coupled with enriched methylated DNA was performed in primary tumors and compared with adjacent nontumor tissues of 12 patients with CRC at stages I to IV. Stepwise filtering for candidate selection in microarray data analysis yielded a set of genes that are highly methylated across all CRC tumors and that can be used as a composite biomarker for CRC detection. Verification assay identified the SDC2 gene as a potential methylation biomarker for early CRC detection. In clinical validation in tissues from 139 CRC patients, a much higher level of aberrant SDC2 methylation was measured in most primary tumors (97.8%), compared with corresponding nontumor tissue of CRC patients, irrespective of clinical stage. Clinical validation of SDC2 methylation in serum DNA from CRC patients (n = 131) at stages I to IV and from healthy individuals (n = 125) by quantitative methylation-specific PCR demonstrated a high sensitivity of 87.0% (95% CI, 80.0% to 92.3%) in detecting cancers, with a specificity of 95.2% (95% CI, 89.8% to 98.2%). Importantly, sensitivity at stage I was 92.3%, indicating the potential of SDC2 methylation as a blood-based DNA test for early detection of CRC.

PMID: 23747112

 

Supplement:

Colorectal cancer (CRC) is the second leading cancer killer in the US, affecting both men and women. However, CRC is curable when diagnosed at an early stage. Furthermore, this disease is preventable if detected at precancerous stages such as polyps because abnormal lesion can be safely removed colonoscopically. When it comes to detecting CRC there are a few screening choices for CRC, including colonoscopy, fecal occult blood testing (FOBT), and fecal immunochemical testing (FIT). Colonoscopy is the gold standard of CRC screening, but patients are often reluctant to undergo the procedure – mostly due to the unpleasant preparation – which has curbed widespread adoption. FOBT and FIT are non-invasive stool tests but have limited sensitivity, particularly for an early stage of the disease. Under these screening programs in 2009, about 137,000 people in the US were diagnosed with CRC, with a 40% mortality rate. There is an urgent need for a more preferable and effective option such as a non-invasive blood test with a sufficient sensitivity and specificity in order to save more lives. Our team has spent the last several years working on this development of the new non-invasive diagnostics that can facilitate the detection of CRC patients at earlier stage. We focused on DNA methylation event, one of the forms of epigenetic modification that may regulate gene activity, without changing parts of the DNA sequence directly. In this study, we identified that abnormal methylation event at regulatory region of syndecan-2 (SDC2) is the most suitable for that aim. We have then successfully developed the abnormal methylation SDC2 as a new biomarker for the non-invasive molecular DNA test that measures its status in blood serum from CRC patient. In search for the methylation biomarker candidate that could be used for the early detection of CRC, we established a comprehensive microarray-based DNA methylation analysis coupled with the enrichment of methylated DNAs providing the differential gene methylation patterns between the tumor and non-tumor tissue cells. After step-wise filtering, we found a set of genes that were highly methylated across all of the CRC tumors on the other hand it showed little or no in non-tumors. Ultimately, we identified one gene, SDC2, which encodes the membrane syndecan-2 protein (a protein that is known to participate in cell proliferation and cell migration) that is expressed in colon mesenchymal cells. The methylation level of target region of SDC2 was verified by a different methylation assay, bisulfite pyro-sequencing, to be significantly higher in the tumors than paired adjacent non-tumor tissues. This phenomenon was further clinically validated in a different sample set of primary tumors and paired-adjacent non-tumor tissue from 133 CRC patients. Our goal was to detect aberrant methylation of SDC2 in a circulating cell-free DNA in the blood. Therefore, we first established a quantitative methylation specific PCR (qMSP) using real-time PCR which can measure a minute amount of target DNA, enabling to measure the SDC2 biomarker in serum samples from CRC patients and healthy individuals. The qMSP-SDC2 methylation test was able to detect 114 cancer patients out of 131 patients with various CRC stages (sensitivity 87%) while correctly identifying 95% of disease-free patients (specificity). Remarkably, this test was also able to detect 92% of stage I cancer patients indicating that this biomarker is suitable for early detection of CRC when therapeutic interventions have the greatest likelihood of curing the patient. After our previous report, we have made an effort to improve the accuracy of this non-invasive test in order to detect abnormal SDC2 methylation in the serum derived from patient with precancerous lesion like a polyp. As result, our current serum-based qMSP test comprised 1) improved DNA purification steps using magnetic bead using a larger volume of blood which allows obtaining at least 1ml of serum and 2) optimized degenerated primer set for PCR. We are currently performing another set of clinical validation studies which evaluates SDC2 methylation statue quantitatively in serum DNA derived from patients having polyps. We were able to see a high potential of a new non-invasive blood test in detecting not only patients with early stage CRC but also precancerous lesion with high sensitivity (Results will be poster-presented at the Molecular Medicine Tri- Conference 2014 San Francisco). Furthermore, we found that the level of SDC2 methylation in serum DNA from CRC patients dramatically dropped to a basal level after curative surgery, indicating that it could also be used to monitor cancer progression and treatment. In the future, we will explore whether this biomarker is universal among other cancers and could possibly be used in conjunction with/or an alternative to colonoscopy.

Sungwhan-fig1

Grant support: This study was supported by a grant from Ministry for Health, Welfare and Family affairs (Project No. 0405-BC01-0604-0002), South Korea.

Contact:

Sungwhan An, Ph.D., CEO/CSO at Genomictree, Inc.

Address: 829 Tamnip-dong, Yuseong-gu, Daejeon, 305-510, South Korea.

Email: genomictree1@korea.com. Phone: +82-42-861-4550. Fax: +82-42-861-4552

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