Abstract #1326: A genome-wide screen for DNA methylation-based colorectal cancer biomarkers: Discovery of new candidates and search for complementarity to Septin 9.
Authors: Shannon Payne, Cathy Lofton-Day, Fabian Model, Theo DeVos, Volker Liebenberg, Reimo Tetzner, Juergen Distler, Matthias Schuster, Andrew Sledziewski
Journal: Cancer Research (2009)

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite a survival rate of over 90% when detected early. Currently, less than half of guideline eligible individuals are screened regularly for CRC, highlighting a need for improved non-invasive methods of early detection. We previously identified and validated a blood-based biomarker, Septin 9, which identifies methylated DNA in plasma from all stages of CRC. We have now conducted a genome-wide screen to identify additional blood-based biomarkers that either augment the performance of Septin 9 for CRC detection or perform individually as well as Septin 9. Methods: Differential methylation hybridization (DMH) via customized high-density microarrays with >50,000 features was used to explore methylation differences between Septin 9 (-) CRC (n = 13), Septin 9 (+) CRC (n = 14), normal adjacent to tumor (NAT, n = 11) and whole blood (n = 8) DNA. Significant probesets were identified using a Benjamin-Hochberg false discovery rate of 0.25 and ranked by t statistic. The top 65 DMH and 62 literature candidates were chosen for real-time PCR assay development and validated using tissues from 51 Septin 9 (-) CRCs, 147 Septin 9 (+) CRCs, 8 NATs and 14 normal colon epithelia in addition to 94 other disease and normal tissues. Ten of these candidates were chosen for HMO assay development and the performances of the four most promising candidates were evaluated in plasma from 136 CRC patients and 170 colonoscopy-verified controls using a case-control study design. Results: 7026 statistically significant probesets for all CRC tissues versus NAT and whole blood and 7280 significant probesets for Septin 9 (+) CRC versus Septin 9 (-) CRC were identified. Comparison of the top 150 candidates for Septin 9 (-) CRC versus whole blood and versus Septin 9 (+) CRC yielded 224 unique candidates. Results of real-time PCR analysis validated 26 of 45 biomarker candidates, with significant overlap between those CRC tissues identified by Septin 9 and those identified by the new candidates (all Spearman R values > 0.48). Using HM real-time PCR analysis in DNA extracted from plasma, Septin 9 detected 85/136 CRC patients (63%) and 27/170 controls (16%). The four new biomarkers detected from 54/136 CRC patients (40%) to 74/136 CRC patients (54%) and from 18/170 controls (11%) to 42/170 controls (25%). Conclusions: By using a systematic screen, we identified 26 biomarkers that specifically discriminated CRC from normal colon tissues, although complementarity to Septin 9 was limited. Plasma-based, highly sensitive real-time PCR assays were successfully developed for four of these biomarkers. All biomarkers identified a similar subset of CRC patient plasmas, with slight variations in sensitivity and specificity. Septin 9 remains the best biomarker for maximizing sensitivity and specificity on plasma DNA.  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1326.