Novel methylation biomarker panel for the early detection of pancreatic cancer.

PURPOSE: Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here, we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer. EXPERIMENTAL DESIGN: We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines. Of eight promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis, including methylation and expression. We used a nanoparticle-enabled methylation on beads (MOB) technology to detect early-stage pancreatic cancers by analyzing DNA methylation in patient serum. RESULTS: We identified two novel genes, BNC1 (92%) and ADAMTS1 (68%), that showed a high frequency of methylation in pancreatic cancers (n = 143), up to 100% in PanIN-3 and 97% in stage I invasive cancers. Using the nanoparticle-enabled MOB technology, these alterations could be detected in serum samples (n = 42) from patients with pancreatic cancer, with a sensitivity for BNC1 of 79% [95% confidence interval (CI), 66%-91%] and for ADAMTS1 of 48% (95% CI, 33%-63%), whereas specificity was 89% for BNC1 (95% CI, 76%-100%) and 92% for ADAMTS1 (95% CI, 82%-100%). Overall sensitivity using both markers is 81% (95% CI, 69%-93%) and specificity is 85% (95% CI, 71%-99%). CONCLUSIONS: Promoter DNA methylation of BNC1 and ADAMTS1 is a potential biomarker to detect early-stage pancreatic cancers. Assaying the promoter methylation status of these genes in circulating DNA from serum is a promising strategy for early detection of pancreatic cancer and has the potential to improve mortality from this disease.

DNA methylation detection using MS-qFRET, a quantum dot-based nanoassay.

Detection of aberrant promoter hypermethylation of tumor suppressor genes can be used as a prognostic or predictive marker for carcinogenesis. Since epigenetic modifying agents are FDA approved for treatment of patients with myelodysplastic syndrome, laboratory correlative tools to monitor response to this targeted therapy are important. Methylation specific quantum dot fluorescence resonance energy transfer (MS-qFRET) is a nanotechnology assay that enables the detection of methylation and its changes in a sensitive, quantifiable manner. It utilizes quantum dot-mediated fluorescence resonance energy transfer to achieve highly sensitive detection of DNA methylation. Template DNA is first treated with sodium bisulfite such that unmethylated cytosines are converted to uracil while methylated cytosines remain unconverted. Thereafter, the converted template is amplified using biotinylated methylation-specific primers. Quantum dots, functionalized with streptavidin, serve both as a scaffold to capture amplicons and as a donor for transferring energy to the Cy5 acceptor that is incorporated into the amplicons during PCR. Thus, the status of DNA methylation can be determined according to the level of FRET. In this report, MS-qFRET is validated in cell lines and then used to detect the status of p15(INK4B) methylation in clinical samples from eight patients with acute myeloid leukemia.

Single-tube analysis of DNA methylation with silica superparamagnetic beads.

BACKGROUND: DNA promoter methylation is a signature for the silencing of tumor suppressor genes. Most widely used methods to detect DNA methylation involve 3 separate, independent processes: DNA extraction, bisulfite conversion, and methylation detection via a PCR method, such as methylation-specific PCR (MSP). This method includes many disconnected steps with associated losses of material, potentially reducing the analytical sensitivity required for analysis of challenging clinical samples. METHODS: Methylation on beads (MOB) is a new technique that integrates DNA extraction, bisulfite conversion, and PCR in a single tube via the use of silica superparamagnetic beads (SSBs) as a common DNA carrier for facilitating cell debris removal and buffer exchange throughout the entire process. In addition, PCR buffer is used to directly elute bisulfite-treated DNA from SSBs for subsequent target amplifications. The diagnostic sensitivity of MOB was evaluated by methylation analysis of the CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); also known as p16(INK4a)] promoter in serum DNA of lung cancer patients and compared with that of conventional methods. RESULTS: Methylation analysis consisting of DNA extraction followed by bisulfite conversion and MSP was successfully carried out within 9 h in a single tube. The median pre-PCR DNA yield was 6.61-fold higher with the MOB technique than with conventional techniques. Furthermore, MOB increased the diagnostic sensitivity in our analysis of the CDKN2A promoter in patient serum by successfully detecting methylation in 74% of cancer patients, vs the 45% detection rate obtained with conventional techniques. CONCLUSIONS: The MOB technique successfully combined 3 processes into a single tube, thereby allowing ease in handling and an increased detection throughput. The increased pre-PCR yield in MOB allowed efficient, diagnostically sensitive methylation detection.