A simplified, noninvasive stool DNA test for colorectal cancer detection.

BACKGROUND: As a noninvasive colorectal cancer (CRC) screening test, a multi-marker first generation stool DNA (sDNA V 1.0) test is superior to guaiac-based fecal occult blood tests. An improved sDNA assay (version 2), utilizing only two markers, hypermethylated vimentin gene (hV) and a two site DNA integrity assay (DY), demonstrated in a training set (phase 1a) an even higher sensitivity (88%) for CRC with a specificity of 82%. AIM: To validate in an independent set of patients (phase 1b) the sensitivity and specificity of sDNA version 2 for CRC. METHODS: Forty-two patients with CRC and 241 subjects with normal colonoscopy (NC) provided stool samples, to which they immediately added DNA stabilizing buffer, and mailed their specimen to the laboratory. DNA was purified using gel-based capture, and analyzed for hV and DY using methods identical to those previously published. RESULTS: Using the same cutpoints as the 1a training set (N = 162; 40 CRCs, 122 normals), hV demonstrated a higher and DY a slightly lower sensitivity, for a combined sensitivity of hV + DY of 86%. Optimal cutpoints based on the combined phase 1a + 1b dataset (N = 445; 82 CRCs, 363 normals) yielded a CRC sensitivity of 83%. The vast majority of cancers were detected regardless of tumor stage, tumor location, or patient age. Assay specificity in the phase 1b dataset for hV, DY, and hV + DY was 82%, 85%, and 73%, respectively, using the phase 1a cutpoints. Optimal cutpoints based on the combined phase 1a + 1b dataset yield a specificity of 82%. CONCLUSIONS: This study provides validation of a simplified, improved sDNA test that incorporates only two markers and that demonstrates high sensitivity (83%) and specificity (82%) for CRC. Test performance is highly reproducible in a large set of patients. The use of only two markers will make the test easier to perform, reduce the cost, and facilitate distribution to local laboratories.

Improved fecal DNA test for colorectal cancer screening.

BACKGROUND & AIMS: Fecal DNA testing has shown greater sensitivity than guaiac-based occult blood tests for noninvasive colorectal cancer (CRC) screening. The prototype assay (version 1), which analyzed 22 gene mutations and DNA integrity assay (DIA), showed a sensitivity of 52% for CRC detection and a specificity of 94% in average-risk individuals. The present study was conducted to determine the sensitivity and specificity of a second-generation assay (version 2) that uses improved DNA stabilization/isolation techniques and a new promoter methylation marker. METHODS: Forty patients with CRC and 122 subjects with normal colonoscopy provided stool samples to which DNA preservation buffer was added immediately. DNA was purified using gel-based capture, and analyzed for the original panel of 22 mutations, DIA, and 2 new promoter methylation markers. RESULTS: By using DNA that was optimally preserved and purified from stool, the sensitivity of the prototype version 1 assay increased to 72.5% because of enhanced performance of DIA. Vimentin gene methylation alone provided sensitivity and specificity of 72.5% and 86.9%, respectively. The optimal combination of vimentin methylation plus DIA resulted in 87.5% sensitivity and 82% specificity; cancers were detected regardless of stage or location. False-positive vimentin methylation was associated with older age. CONCLUSIONS: An improved fecal DNA test that incorporates only 2 markers shows much higher sensitivity for CRC. The new assay is easier to perform and should be less costly, thereby facilitating its use for noninvasive CRC screening.

Sensitive digital quantification of DNA methylation in clinical samples.

Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers. We have developed methyl-BEAMing technology to enable absolute quantification of the number of methylated molecules in a sample. Individual DNA fragments are amplified and analyzed either by flow cytometry or next-generation sequencing. We demonstrate enumeration of as few as one methylated molecule in approximately 5,000 unmethylated molecules in DNA from plasma or fecal samples. Using methylated vimentin as a biomarker in plasma samples, methyl-BEAMing detected 59% of cancer cases. In early-stage colorectal cancers, this sensitivity was four times more than that obtained by assaying serum-carcinoembryonic antigen (CEA). With stool samples, methyl-BEAMing detected 41% of cancers and 45% of advanced adenomas. In addition to diagnostic and prognostic applications, this digital quantification of rare methylation events should be applicable to preclinical assessment of new epigenetic biomarkers and quantitative analyses in epigenetic research.