Validation of a Real-Time PCR–Based Qualitative Assay for the Detection of Methylated SEPT9 DNA in Human Plasma

BACKGROUND Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test. METHODS Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n = 1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard. RESULTS The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6–11 pg/mL) corresponding to <2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%–80%) and for stage I–III CRC, 64% (48%–77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%–82%). SIGNIFICANCE: The Epi proColon test is a simple, real-time PCR–based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines.