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 8 promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis including methylation and expression. We used a nanoparticle-enabled MOB (Methylation On Beads) technology to detect early stage pancreatic cancers by analyzing DNA methylation in patient serum. Results We identified 2 novel genes, BNC1 (92%) and ADAMTS1, (68%) that showed a high frequency of methylation in pancreas 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 pancreas cancer patients, with a sensitivity for BNC1 of 79% (95%CI:66-91%) and for ADAMTS1 of 48% (95%CI:33-63%), while 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 are potential biomarkers 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 contributes to carcinogenesis by silencing key tumor suppressor genes. Here we report an ultrasensitive and reliable nanotechnology assay, MS-qFRET, for detection and quantification of DNA methylation. Bisulfite-modified DNA is subjected to PCR amplification with primers that would differentiate between methylated and unmethylated DNA. Quantum dots are then used to capture PCR amplicons and determine the methylation status via fluorescence resonance energy transfer (FRET). Key features of MS-qFRET include its low intrinsic background noise, high resolution, and high sensitivity. This approach detects as little as 15 pg of methylated DNA in the presence of a 10,000-fold excess of unmethylated alleles, enables reduced use of PCR (as low as eight cycles), and allows for multiplexed analyses. The high sensitivity of MS-qFRET enables one-step detection of methylation at PYCARD, CDKN2B, and CDKN2A genes in patient sputum samples that contain low concentrations of methylated DNA, which normally would require a nested PCR approach. The direct application of MS-qFRET on clinical samples offers great promise for its translational use in early cancer diagnosis, prognostic assessment of tumor behavior, as well as monitoring response to therapeutic agents.[Supplemental material is available online at www.genome.org.]Aberrant DNA hypermethylation is observed at classic tumorsuppressor genes, which are known to be genetically mutated and cause inherited forms of cancer (Jones and Baylin 2002). Tumor cells display a larger number of genes inactivated by promoter hypermethylation than by genetic mutations (Schuebel et al. 2007). Furthermore, these abnormal epigenetic changes appear to be an early event that precedes detection of genetic mutations (Esteller et al. 1999;Feinberg and Tycko 2004;Yamada et al. 2005). Thus, detection of promoter hypermethylation is a valuable tool for early diagnosis of cancer, monitoring tumor behavior, as well as measuring response of tumors to targeted therapy (Esteller et al. 2000;Gore et al. 2006b;Brock et al. 2008).The number of tools available to assess DNA methylation demonstrates the extensive interest that has been invested in understanding the role of epigenetics in carcinogenesis (Laird 2003). One of the more common techniques used for the detection of methylation is methylation-specific PCR (MSP) (Herman et al. 1996). The technique relies on sodium bisulfite treatment of DNA, which converts unmethylated cytosines to uracils while leaving methylated cytosines unaffected. The modified sequences are then amplified with specific primers, and the amplified products are identified using gel electrophoresis. However, this standard MSP approach offers only qualitative analysis and cannot discern relative amounts of methylation. Although real-time PCRbased MSP methods (Lo et al. 1999;Eads et al. 2000) enable quantitative analysis, they may lack the sensitivity for direct screening of challenging samples, such as sputum, where the DNA from tumor cells is minimal, thereby requiring a...
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 p16INK4a] 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.
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