Alejandro Stark

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

Early Detection of Lung Cancer Using DNA Promoter Hypermethylation in Plasma and Sputum.

Purpose: CT screening can reduce death from lung cancer. We sought to improve the diagnostic accuracy of lung cancer screening using ultrasensitive methods and a lung cancer–specific gene panel to detect DNA methylation in sputum and plasma. Experimental Design: This is a case–control study of subjects with suspicious nodules on CT imaging. Plasma and sputum were obtained preoperatively. Cases (n = 150) had pathologic confirmation of node-negative (stages I and IIA) non–small cell lung cancer. Controls (n = 60) had non-cancer diagnoses. We detected promoter methylation using quantitative methylation-specific real-time PCR and methylation-on-beads for cancer-specific genes (SOX17, TAC1, HOXA7, CDO1, HOXA9, and ZFP42). Results: DNA methylation was detected in plasma and sputum more frequently in people with cancer compared with controls (P < 0.001) for five of six genes. The sensitivity and specificity for lung cancer diagnosis using the best individual genes was 63% to 86% and 75% to 92% in sputum, respectively, and 65% to 76% and 74% to 84% in plasma, respectively. A three-gene combination of the best individual genes has sensitivity and specificity of 98% and 71% using sputum and 93% and 62% using plasma. Area under the receiver operating curve for this panel was 0.89 [95% confidence interval (CI), 0.80–0.98] in sputum and 0.77 (95% CI, 0.68–0.86) in plasma. Independent blinded random forest prediction models combining gene methylation with clinical information correctly predicted lung cancer in 91% of subjects using sputum detection and 85% of subjects using plasma detection. Conclusions: High diagnostic accuracy for early-stage lung cancer can be obtained using methylated promoter detection in sputum or plasma. Clin Cancer Res; 23(8); 1998–2005. ©2016 AACR.

A parallelized microfluidic DNA bisulfite conversion module for streamlined methylation analysis.

Aberrant methylation of DNA has been identified as an epigenetic biomarker for numerous cancer types. The vast majority of techniques aimed at detecting methylation require bisulfite conversion of the DNA sample prior to analysis, which until now has been a benchtop process. Although microfluidics has potential benefits of simplified operation, sample and reagent economy, and scalability, bisulfite conversion has yet to be implemented in this format. Here, we present a novel droplet microfluidic design that facilitates rapid bisulfite conversion by reducing the necessary processing steps while retaining comparable performance to existing methods. This new format has a reduced overall processing time and is readily scalable for use in high throughput DNA methylation analysis.

A new immunohistochemistry prognostic score (IPS) for recurrence and survival in resected pancreatic neuroendocrine tumors (PanNET)

Pancreatic neuroendocrine tumor (PanNET) is a neoplastic entity in which few prognostic factors are well-known. Here, we aimed to evaluate the prognostic significance of N-myc downstream-regulated gen-1 (NDRG-1), O6-methylguanine DNA methyltransferase (MGMT) and Pleckstrin homology-like domain family A member 3 (PHLDA-3) by immunohistochemistry (IHC) and methylation analysis in 92 patients with resected PanNET and follow-up longer than 24 months. In multivariate analyses, ki-67 and our immunohistochemistry prognostic score (IPS-based on MGMT, NDRG-1 and PHLDA-3 IHC expression) were independent prognostic factors for disease-free-survival (DFS), while age and IPS were independent prognostic factors for overall survival (OS). Our IPS could be a useful prognostic biomarker for recurrence and survival in patients following resection for PanNET.

The promise of methylation on beads for cancer detection and treatment

Despite numerous technical hurdles, the realization of true personalized medicine is becoming a progressive reality for the future of patient care. With the development of new techniques and tools to measure the genetic signature of tumors, biomarkers are increasingly being used to detect occult tumors, determine the choice of treatment and predict outcomes. Methylation of CpG islands at the promoter region of genes is a particularly exciting biomarker as it is cancer-specific. Older methods to detect methylation were cumbersome, operator-dependent and required large amounts of DNA. However, a newer technique called methylation on beads has resulted in a more uniform, streamlined and efficient assay. Furthermore, methylation on beads permits the extraction and processing of miniscule amounts of methylated tumor DNA in the peripheral blood. Such a technique may aid in the clinical detection and treatment of cancers in the future.

Quantum dot FRET linker probes for highly sensitive DNA methylation detection

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.

A sample-to-answer droplet magnetofluidic assay platform for quantitative methylation-specific PCR

Dysregulation of DNA methylation has been identified as an epigenetic biomarker for numerous cancer types. Gene-specific identification techniques relying on methylation-specific PCR (MSP) require a lengthy manual benchtop process that is susceptible to human-error and contamination. This MSP assay requires a series of discrete sample processing steps including genomic DNA extraction, bisulfite conversion and readout via PCR. In this work, we present a streamlined assay platform utilizing droplet magnetofluidic principles for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples. We present a streamlined protocol for solid-phase extraction and bisulfite conversion of genomic DNA, which minimizes reagent use and simplifies the sample preparation protocol for implementation on a compact assay platform. Furthermore, we present a thermally robust assay chip that enables DNA extraction, bisulfite conversion and quantitative PCR from biological samples on a single device. Technical improvements to facilitate DNA extraction and PCR on a single chip in addition to chip performance characterization data are presented.

Increasing throughput and sensitivity of DNA Methylation analysis through functional nanoparticles

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.

DNA methylation density analysis via fluorescent dye incorporation

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.