- 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

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.

Abstract Pancreatic cancer is a deadly cancer with an overall 5-year survival rate of less than 5% and no improvements in survival over the last 3 decades. Pancreatic cancer currently ranks as the fourth leading cause of cancer related death in United States with an estimated 42,470 new cases and 35,240 deaths in 2009 and its incidence is rising. One of the major factors attributed to the dismal prognosis of pancreas cancer is their delayed diagnosis such that only about 10% cases are amenable to potential curative surgical resection. However, long term 5-year survival is attainable in selected patients with early-stage pancreatic cancer who can undergo curative surgical resection. Early detection of pancreatic cancer is, therefore, thought to be the best modality for improving survival in this lethal disease. However, no screening test currently exists for pancreatic cancer. In recent years, it has become apparent that pancreatic cancer is as much a disease of mis-regulated epigenetics as it is a disease of genetic mutation. In particular, changes in DNA promoter methylation patterns could play a crucial role in tumorigenesis and cancer progression. In order to address the need for both clinical diagnostics as well as therapeutics, many studies have employed DNA methylation of specific genes for application in diagnostics of multiple cancers. Detection of cancer specific, abnormally DNA methylated gene promoter sequences has emerged as one of the leading tumor, biomarker detection strategies. We have used a genome-wide transcriptome approach to identify new cancer specific DNA methylation alteration in pancreatic carcinoma. We analyzed methylation frequencies of best candidate genes, BNC1 and ADAMTS1, by MSP and qMSP as well as expression analysis by real-time PCR and immunohistochemistry. We use a novel nanoparticle-enabled MOB (Methylation On Beads) technology to detect very early stages of the pancreatic cancers. The biological role of BNC1 gene was examined by colony formation, cell proliferation, and invasion assays in pancreatic cancer cell lines. We identified 2 novel genes BNC1 (91.8%) and ADAMTS1 (66.7%) that showed a high frequency of methylation in pancreas cancer tissues (n=143). BNC1 was frequently methylated in the earliest stages of pancreas carcinogenesis including carcinoma in situ or pancreatic intraepithelial neoplasia PanIN3 (100%) and Stage 1 invasive cancers (97.4%). Using the ultrasensitive nanoparticle-enabled MOB assay, these alterations could be detected in serum samples from patients with pancreas cancer, with a sensitivity for BNC1 of 79% (95% CI: 0.6–0.8) and for ADAMTS1 of 48% (95% CI: 0.3–0.6) (n=42 cancers, Stages 1–4), while specificity was 88% for BNC1 (95% CI: 0.6–0.9) and 92% for ADAMTS1 (95% CI: 0.7–0.9) among 26 individuals without cancer. BNC1 overexpresstion in pancreatic cancer cell lines showed suppressive effect by colony formation, cell proliferation but not invasion. Both BNC1 and ADAMTS1 had high sensitivity for the earliest stages of pancreas cancers. Notably, BNC1 and ADAMTS1 show the potential power of using circulating DNA for early detection of cancer, especially in high risk individuals. Moreover, BNC1 is a candidate tumor suppressor gene in pancreatic cancer which is inactivated by promoter DNA methylation. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B43.

Genomic analysis of biomarkers, including genetic markers such as point mutations and epigenetic markers such as DNA methylation, has become a central theme in modern disease diagnosis and prognosis. Recently there is an increasing interest in using single-molecule detection (SMD) for genomic detection. The driving force not only comes from its ultrahigh sensitivity that can allow the detection of low-abundance nucleic acids with reduced or without the need of amplification but also from its potential in achieving high-accuracy quantification of rare targets via singlemolecule sorting. The unique photophysical properties of semiconductor quantum dots (QDs) have made them ideal for use as spectral labels and luminescent probes. QDs also make excellent donors to pair with organic dyes in the fluorescence resonance energy transfer (FRET) process due to the features of narrow emission spectra and small Stokes shift. We have developed highly sensitive, quantitative and clinically relevant technologies for analysis of genomic markers based on the convergence of SMD, microfluidic manipulations, and quantum dot fluorescence resonance energy transfer technology (QD-FRET). Extraordinary performances of these new technologies have been exemplified by analysis of a variety of biomarkers including point mutations, DNA integrity and DNA methylation in clinical samples.

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.

Background: One of the often occurred epigenetic events associated to cancer is inheritable transcriptional silencing of the tumor suppressor genes by aberrant methylation of cytosines at the promoter regions. Current approaches to detect promoter DNA methylation commonly involve three separate independent processes, including DNA extraction, bisulfite conversion and methylation detection through PCR amplification, such as methylation specific PCR (MSP). This method includes many disconnected steps with loss of genetic materials, potentially reducing the sensitivity required for analysis of challenging clinical samples. Methods and Results: To address this problem, we developed a new technique named Methylation-on-Beads (MOB). MOB uses the silica superparamagnetic nanobeads (SSB) to combine DNA extraction, bisulfite treatment (Bst) and PCR into a single tube. The single-tube scheme minimizes the DNA loss during the tube transfers and improves the analytical sensitivity for methylation detection. Further enhancement in sensitivity is achieved by combining MOB with quantum dot-enhanced MSP detection. The pre-PCR DNA yields using MOB were compared with those carried out with conventional organic solvent extraction and ethanol precipitation (PC). 15 serum samples from lung cancer patients (7 Stage I, 3 Stage II, 5 Stage III) were analyzed using both MOB and PC. The DNA recovery using MOB was higher than PC for each patient serum sample with a median increase of 6.61 fold. To demonstrate the versatile applicability of the new method, we further analyzed 10 samples to include fresh tissue and paraffin embedded tissue from normal patients, fresh tumors from cancer patients and sputum samples. Median DNA yield increase of 7.8, 5.3, 6.4 and 7.5 respectively was determined using MOB when compared to the conventional method. We examined the outcome of bisulfite conversion in the presence of SSBs using real-time methylation specific PCR (MSP) to analyze p16INK4a promoter methylation. A set of triplicate reactions were examined with bisulfite-treated DNA of varying treatment durations (0h, 1h, 3h, 4h, 8h), and compared to the control using 16 hrs of conventional bisulfite treatment. Results indicated that 4h of bisulfite treatment was sufficient for conversion, and that the presence of beads did not alter the conversion process. Assessment of methylation in serum or plasma can be a useful tool for early detection of cancer. However, extending the DNA methylation analysis to clinically usable serum/ blood-based tests has been limited by the lack of sensitivity of conventional methods. In order to address whether improving DNA yields could enhance methylation detection, we compared methylation of p16INK4a promoter in 49 patient serum samples (18 normal and 31 cancer) in a blinded study using both MOB and PC/Bst/MSP. The 31 tumor samples were pre-selected from patients diagnosed with lung cancer who were also methylated for p16INK4a promoter in corresponding tumors. While p16INK4a methylation was detected in 14/31 patients with lung cancer using conventional approach; using MOB, we were able to detect p16INK4a methylation in 23/31of these patients. When samples used for methylation analysis contained large amounts of DNA (cell lines, tumors etc), a single-tube analysis of entire input amount may be unnecessary. Instead, multiple reactions in parallel were feasible by directly splitting the SSB into several different tubes. Conclusion: MOB successfully combined three processes required for DNA methylation analysis into a single-tube using SSB thereby allowing for ease in handling and increased throughput in detection. Increased pre-PCR yield in MOB allowed for efficient, diagnostically sensitive methylation detection.

We performed on-chip DNA methylation analysis using methylation-specific PCR (MSP) within a high throughput microfluidic droplet array. The device uses of the oil phase as a companion fluid for both sample actuation and compartmentalization. These technical advantages allow for infusion of minute amounts of sample for arrayed MSP analysis, without the added complexities inherent in microfluidic droplet-based studies. Ease of use of this micro device is exemplified by analysis of two tumor suppressor promoters, p15 and TMS1 using an on-chip methylation assay. These results were consistent with standard MSP protocols, yet the simplicity of the droplet-in-oil microfluidic PCR platform provides an easy and efficient tool for DNA methylation analysis in a large-scale arrayed manner.