- PDF file 64K, Supplementary Table S1. Primers used for MSP, RT-PCR, Bisulfite sequencing analyses

- PDF file 86K, Supplementary Table S2. BNC1 and ADAMTS1 Gene expression changes in pancreatic cancers from previously published microarray dataset

- PDF file 93K, This includes figure legends of supplementary Figure S1-S3

- PDF file 108K, Supplementary Figure S2. mRNA and Protein Expression in BNC1 Overexpression System

- PDF file 69K, Supplementary Fig. S1. Schematic representation of the study design used in this project

- PDF file 57K, Supplemental Figure S3. Kaplan-Meier Curve of Overall Survival Characterized by ADAMTS1 Methylation

384 Background: Identification of blood-based biomarkers for cancer screening is essential in order to develop novel and minimally invasive methods for colorectal cancer screening. Our lab has successfully applied a novel nanotechnology that allows us to detect and amplify a single tumor DNA fragment in a plasma sample. This DNA is tested for methylation of several genes including TFPI2 which has shown to be highly sensitive and specific for the detection colorectal cancer in stool. Methods: Whole blood was obtained from 18 colorectal cancer patients and plasma was isolated. Plasma was processed using Methylation On Beads nanotechnology (MOB) and bisulfate treated. Methylation status was determined via quantitative PCR method. Results: Two genes, TFPI2 and IGFBP3, were detected with a high sensitivity. TFPI2, demonstrated a methylation frequency of 94.4%, which is concordant with the TFPI2 methylation frequency of 99% in primary colorectal cancer tissues. IGFBP3 showed the methylation frequency of 61.1%, which corresponds with the methylation frequency of 52% in retrospective colorectal cancer tissues in previous studies. Quantification using standard curves indicated a single copy level of DNA found in plasma. Conclusions: Blood-based screening is challenging due to extremely low quantities of circulating DNA in blood. Utilizing a novel nanotechnology that detects DNA at a single copy level, the methylation changes in colorectal cancer were successfully detected in plasmas at similar frequencies as in tissue samples. This study has demonstrated the feasablility and applicability to blood-based screening. Future studies will focus on improving the sensitivity and determining the specificity of this method.

Abstract 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. Clin Cancer Res; 19(23); 6544–55. ©2013 AACR.

The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed "methylation on beads" that allows for DNA extraction and bisulfite conversion for up to 2 ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5- to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays.

DNA methylation is an important cancer biomarker, but improving the detection sensitivity of DNA methylation remains a challenge for various types of biological samples. Here, we describe a novel quantum dot (QD)-based method using FRET linker probes (FLPs) that further increases the sensitivity of detection of DNA methylation patterns. This method relies on removing the background noise resulting from PCR inefficiencies. Compared to conventional non-specific QD-FRET detection, we demonstrate the improved detection of DNA methylation by fluorescence detection in bulk and with single molecule resolution.

DNA Methylation analysis has been proven as an invaluable tool in cancer screening and diagnosis. Conventional techniques for DNA methylation analysis have limited sensitivity and specificity, making early detection a complicated endeavor. Furthermore, DNA methylation analysis requires a series of disconnected processes. Although a wide variety of commercial kits are available for the individual steps, so far there is no product that can combine all the steps together. Our novel approach address these problems by integrating all the steps required for DNA methylation together through the use of the silica superparamagnetic nanoparticles (SSNP) and quantum dots, thus minimizing the sample transfer and reducing the processing time.

Come closer to the light: Fluorescently labeled cytosines have been enzymatically incorporated within bisulfite-treated DNA that was amplified by using methylation-specific primers. Detection of these multilabeled products was carried out by using quantum-dot FRET (QD-FRET). Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Abstract Background: Aberrant DNA methylation of cytosine nucleotides within the promoter region of tumor suppressor genes provides an epigenetic mechanism of transcriptional control associated with carcinogenesis. Thus, DNA methylation analysis holds clinical potential for early detection and treatment of cancer. Both qualitative detection methods such as methylation specific PCR (MSP), and quantitative methods like MethyLight and Ms-SNuPE analyze a proportion of alleles only when they are heavily methylated at primer/probe sequences. Bisulfite sequencing can be useful to understand methylation density but is not often used quantitatively due to the labor intensive sequencing of multiple plasmid clones. Methods and Results: Methylation through Fluorescence of a Single Strand (Methyl-FloSS) establishes a simple detection method for the quantitative analysis of methylation density. Methyl-FloSS begins with the bisulfite treatment of genomic DNA, followed by PCR amplification with primers that are independent of methylation status. Amplification is carried out with labeled nucleotides (Cy5-dCTP) and a 5’ phosphate conjugated primer. Enzymatic digestion is then directed toward the reverse strand containing the phosphate primer, allowing investigation of the remaining strand wherein Cy5 fluorophores are incorporated into the positions of methylated cytosines. Therefore, the methylation density is proportional to the measured fluorescence intensity of the single stranded amplicon. A fluorescence density score (FDS) is calculated to quantify the methylation density. By including a positive and negative control and assigning a FDS of 1 and 0 respectfully, each sample receives a FDS score based upon the normalized fluorescence intensity and DNA concentration. To demonstrate the applicability of Methyl-FloSS, we observed the in vitro effect of 5-aza-2’-deoxycytidine (DAC) on the methylated p15INK4B promoter region in KG-1a cell line. Cells were harvested after 72 hours of incubation with 0 nM, 10 nM, 100 nM, and 1 µM of DAC. Our results demonstrated a dose-dependent decrease in p15INK4B promoter methylation density as measured by decreasing FDS, which is consistent with prior MSP analysis. Demonstrating the clinical significance of the Methyl-FloSS, blood samples from 24 acute myeloid leukemia (AML) patients were comparatively analyzed by both MSP categorization and density analysis. One patient was considered methylated for p15INK4B by MSP, but density analysis measured a low FDS. This result indicates that the presence of methylated cytosines located within the primer sequences of MSP may categorize the patient as methylated; however, the methylation density of the promoter region is comparable with unmethylated controls. Overall, FDS scores allowed for a clear segregation between methylated and unmethylated samples, featuring greater resolution by fluorescence measurement. Conclusion: The enhanced detection of methylation density by Methyl-FloSS provides a convenient, quantitative analysis of promoter methylation status and is not biased by primer/probe sequences. Quantification through FDS will provide greater insight and analysis into epigenetic alterations and therapies. In addition, Methyl-FloSS avoids radioactive labeling and can be easily implemented in a multi-well format for high throughput analysis.

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.

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 p15INK4B methylation in clinical samples from eight patients with acute myeloid leukemia.