Blood Plasma Methylated DNA Markers in the Detection of Lymphoma: Discovery, Validation, and Clinical Pilot.

Lymphoma is one of the leading causes of cancer and cancer deaths and yet has not been amenable to population screening. The role of methylated DNA markers (MDMs) in the detection of lymphoma has not been extensively studied. We aimed to discover, validate, and test tissue-derived MDMs of lymphoma in archival plasma specimens.

Methylated DNA Markers in Voided Urine for the Identification of Clinically Significant Prostate Cancer.

Introduction: Non-invasive assays are needed to better discriminate patients with prostate cancer (PCa) to avoid over-treatment of indolent disease. We analyzed 14 methylated DNA markers (MDMs) from urine samples of patients with biopsy-proven PCa relative to healthy controls and further studied discrimination of clinically significant PCa (csPCa) from healthy controls and Gleason 6 cancers.

Methods: To evaluate the panel, urine from 24 healthy male volunteers with no clinical suspicion for PCa and 24 men with biopsy-confirmed disease across all Gleason scores was collected.

Plasma Assay of Cell-Free Methylated DNA Markers of Colorectal Cancer: A Tumor-Agnostic Approach to Monitor Recurrence and Response to Anticancer Therapies.

Background: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies.

Plasma Methylated DNA Markers for Melanoma Surveillance.

Purpose: Surveillance after primary melanoma treatment aims to detect early signs of low-volume systemic disease. The current standard of care, surveillance imaging, is costly and difficult to access. We therefore sought to develop methylated DNA markers (MDMs) as promising alternatives for disease surveillance.

Methylated DNA markers for plasma detection of ovarian cancer: Discovery, validation, and clinical feasibility.

Objective: Aberrant DNA methylation is an early event in carcinogenesis which could be leveraged to detect ovarian cancer (OC) in plasma.

Methods: DNA from frozen OC tissues, benign fallopian tube epithelium (FTE), and buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify OC MDMs. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls.

Detection of VAR2CSA-Captured Colorectal Cancer Cells from Blood Samples by Real-Time Reverse Transcription PCR.

Analysis of circulating tumor cells (CTCs) from blood samples provides a non-invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon-related mRNA transcripts (USH1C and CKMT1A).

Validation of a methylated DNA marker panel for the nonendoscopic detection of Barrett's esophagus in a multisite case-control study.

Background And Aims: We previously identified a 5 methylated DNA marker (MDM) panel for the detection of nonendoscopic Barrett's esophagus (BE). In this study, we aimed to recalibrate the performance of the 5 MDM panel using a simplified assay in a training cohort, validate the panel in an independent test cohort, and explore the accuracy of an MDM panel with only 3 markers.

Methods: Participants were recruited from 3 medical centers.

High Detection Rates of Pancreatic Cancer Across Stages by Plasma Assay of Novel Methylated DNA Markers and CA19-9.

Purpose: We have previously identified tissue methylated DNA markers (MDMs) associated with pancreatic ductal adenocarcinoma (PDAC). In this case-control study, we aimed to assess the diagnostic performance of plasma MDMs for PDAC.

Experimental Design: Thirteen MDMs (, and ) were identified on the basis of selection criteria applied to results of prior tissue experiments and assays were optimized in plasma.

Novel Methylated DNA Markers in the Surveillance of Colorectal Cancer Recurrence.

Purpose: We aimed to assess the concordance of colorectal cancer-associated methylated DNA markers (MDM) in primary and metastatic colorectal cancer for feasibility in detection of distantly recurrent/metastatic colorectal cancer in plasma.

Experimental Design: A panel of previously discovered colorectal cancer-associated MDMs was selected. MDMs from primary and paired metastatic colorectal cancer tissue were assayed with quantitative methylation-specific PCR.

Accurate Nonendoscopic Detection of Barrett's Esophagus by Methylated DNA Markers: A Multisite Case Control Study.

Introduction: Nonendoscopic Barrett's esophagus (BE) screening may help improve esophageal adenocarcinoma outcomes. We previously demonstrated promising accuracy of methylated DNA markers (MDMs) for the nonendoscopic diagnosis of BE using samples obtained from a capsule sponge-on-string (SOS) device. We aimed to assess the accuracy of these MDMs in an independent cohort using a commercial grade assay.

Comparison of Tissue-Based Molecular Markers in Younger versus Older Patients with Colorectal Neoplasia.

Background: Emerging colorectal cancer trends demonstrate increased incidence and mortality in younger populations, prompting consideration of average-risk colorectal cancer screening initiation at age 45 versus 50 years. However, screening test performance characteristics in adults 45-49 years have been minimally described. To inform the biologic rationale for multi-target stool DNA (mt-sDNA) screening in younger patients, we analyzed and compared tissue levels of methylation () and mutation () markers included in the FDA-approved, mt-sDNA assay (Cologuard; Exact Sciences Corporation).

Discovery, Validation, and Application of Novel Methylated DNA Markers for Detection of Esophageal Cancer in Plasma.

Purpose: The burden of esophageal cancer continues to rise, and noninvasive screening tools are needed. Methylated DNA markers (MDM) assayed from plasma show promise in detection of other cancers. For esophageal cancer detection, we aimed to discover and validate MDMs in tissue, and determine their feasibility when assayed from plasma.

Methylated DNA in Pancreatic Juice Distinguishes Patients With Pancreatic Cancer From Controls.

Background & Aims: Precursors of pancreatic cancer arise in the ductal epithelium; markers exfoliated into pancreatic juice might be used to detect high-grade dysplasia (HGD) and cancer. Specific methylated DNA sequences in pancreatic tissue have been associated with adenocarcinoma. We analyzed these methylated DNA markers (MDMs) in pancreatic juice samples from patients with pancreatic ductal adenocarcinomas (PDACs) or intraductal papillary mucinous neoplasms (IPMNs) with HGD (cases), and assessed their ability to discriminate these patients from individuals without dysplasia or with IPMNs with low-grade dysplasia (controls).

Novel Methylated DNA Markers Discriminate Advanced Neoplasia in Pancreatic Cysts: Marker Discovery, Tissue Validation, and Cyst Fluid Testing.

Objectives: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy.

Hepatocellular Carcinoma Detection by Plasma Methylated DNA: Discovery, Phase I Pilot, and Phase II Clinical Validation.

Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues.

Detection of Gastric Cancer with Novel Methylated DNA Markers: Discovery, Tissue Validation, and Pilot Testing in Plasma.

Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls.

Analysis of DNA Methylation at Specific Loci in Stool Samples Detects Colorectal Cancer and High-Grade Dysplasia in Patients With Inflammatory Bowel Disease.

Background & Aims: Patients with inflammatory bowel diseases (IBDs), including ulcerative colitis and Crohn's disease, are at increased risk for colorectal cancer (CRC). Analyses of DNA methylation patterns in stool samples have been reported to detect CRC in patients with IBD. We sought to validate these findings in larger cohorts and assess the accuracy of analysis of DNA methylation patterns in stool for detection of CRC and high-grade dysplasia (HGD) normalized to methylation level at ZDHHC1.

Clinical performance of an automated stool DNA assay for detection of colorectal neoplasia.

Background & Aims: Colorectal cancer (CRC) and advanced precancers can be detected noninvasively by analyses of exfoliated DNA markers and hemoglobin in stool. Practical and cost-effective application of a stool DNA-based (sDNA) test for general CRC screening requires high levels of accuracy and high-capacity throughput. We optimized an automated sDNA assay and evaluated its clinical performance.

Quantification of methylated markers with a multiplex methylation-specific technology.

Background: Aberrantly methylated genes represent important markers for cancer diagnosis. We describe a multiplex detection approach to efficiently quantify these markers for clinical applications such as colorectal cancer screening.

Methods: Quantitative allele-specific real-time target and signal amplification (QuARTS) combines a polymerase-based target amplification with an invasive cleavage-based signal amplification.

Epigenetic down-regulation of the tumor suppressor gene PRDM1/Blimp-1 in diffuse large B cell lymphomas: a potential role of the microRNA let-7.

PRDM1/Blimp-1, a master regulator for B cell terminal differentiation, is a putative tumor suppressor in diffuse large B cell lymphomas (DLBCL). Inactivating mutations of PRDM1 have been previously identified in a subset of nongerminal center B cell-like (GCB) DLBCL. We investigated the presence of alternative mechanisms of down-regulating PRDM1 in a cohort of 25 primary DLBCL and six DLBCL cell lines.

Rapid determination of RNA accessible sites by surface plasmon resonance detection of hybridization to DNA arrays.

RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates.

Detection and differentiation of wild-type and vaccine mutant varicella-zoster viruses using an Invader Plus method.

We report the use of a prototype Invader Plus method (Third Wave Technologies, Inc., Madison, WI) for the qualitative detection of varicella-zoster virus (VZV) and differentiation of wild-type and Oka vaccine VZV. The analytical sensitivity of the VZV Invader Plus reagents is at 10 copies per reaction.

Invader plus method detects herpes simplex virus in cerebrospinal fluid and simultaneously differentiates types 1 and 2.

We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.

Quantitation of microRNAs using a modified Invader assay.

The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells.

Modeling of flap endonuclease interactions with DNA substrate.

Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA.