Novel CRISPR-based sequence specific enrichment methods for target loci and single base mutations.

The programmable sequence specificity of CRISPR has found uses in gene editing and diagnostics. This manuscript describes an additional application of CRISPR through a family of novel DNA enrichment technologies. CAMP (CRISPR Associated Multiplexed PCR) and cCAMP (chimeric CRISPR Associated Multiplexed PCR) utilize the sequence specificity of the Cas9/sgRNA complex to target loci for the ligation of a universal adapter that is used for subsequent amplification.

A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment.

Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications.

Vasodilator-Stimulated Phosphoprotein Biomarkers Are Associated with Invasion and Metastasis in Colorectal Cancer.

Background And Aims: The benefit of adjuvant chemotherapy for stage II colorectal cancer (CRC) patients remains unclear, emphasizing the need for improved prognostic biomarkers to identify patients at risk of metastatic recurrence. To address this unmet clinical need, we examined the expression and phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) in CRC tumor progression. VASP, a processive actin polymerase, promotes the formation of invasive membrane structures leading to extracellular matrix remodeling and tumor invasion.

A noninvasive multianalyte urine-based diagnostic assay for urothelial cancer of the bladder in the evaluation of hematuria.

Objective: To test whether a noninvasive urine-based multianalyte diagnostic readout assay that uses protein and DNA biomarkers can risk stratify patients with hematuria into those who are or are not likely to have bladder cancer and those who should receive standard care.

Patients And Methods: This prospective, observational, multicenter, single-assessment study was conducted between June 12, 2009, and April 15, 2011. Eligible patients presented with hematuria and as part of their evaluation underwent cystoscopy.

Molecular grading of tumors of the upper urothelial tract using FGFR3 mutation status identifies patients with favorable prognosis.

Background: Mutations in FGFR3 have been shown to occur in tumors of the upper urothelial tract and may be indicative of a good prognosis. In bladder tumors, the combination of FGFR3 mutation status and Ki-67 level has been used to define a tumor's molecular grade and predict survival. Pathological evaluation of upper urothelial tumors is currently the best predictor of prognosis, but suffers from variability in pathological assessments.

Noninvasive multianalyte diagnostic assay for monitoring bladder cancer recurrence.

Background: The purpose of this study was to establish the clinical performance of a urine-based assay, called a multianalyte diagnostic readout, in monitoring for bladder cancer recurrence.

Methods: This was a prospective, multicenter, single assessment observational study. The multianalyte diagnostic readout uses a combination of one protein and three DNA biomarkers.

Detection of low frequency FGFR3 mutations in the urine of bladder cancer patients using next-generation deep sequencing.

Biological fluid-based noninvasive biomarker assays for monitoring and diagnosing disease are clinically powerful. A major technical hurdle for developing these assays is the requirement of high analytical sensitivity so that biomarkers present at very low levels can be consistently detected. In the case of biological fluid-based cancer diagnostic assays, sensitivities similar to those of tissue-based assays are difficult to achieve with DNA markers due to the high abundance of normal DNA background present in the sample.

A noninvasive multi-analyte diagnostic assay: combining protein and DNA markers to stratify bladder cancer patients.

Purpose: The authors recently reported the development of a noninvasive diagnostic assay using urinary matrix metalloproteinases (MMPs) as monitors of disease-free status and bladder cancer in high-risk populations. Using an approach called clinical intervention determining diagnostic (CIDD), they identified with high confidence those patients who could be excluded from additional intervention. To maximize performance, MMPs were combined with DNA-based markers and CIDD was applied to a population of patients undergoing monitoring for recurrence.

A novel approach to using matrix metalloproteinases for bladder cancer.

Purpose: Given the steadily growing cancer survivor population, increasing pressure has been placed on more effective clinical approaches and biomarker assays to manage care. For bladder cancer despite the high probability of recurrence the number of patients with recurrent disease is significantly lower than the number that remains cancer free at any monitoring interval. We developed a noninvasive urine assay using a novel approach to identify patients without recurrent cancer with extremely high confidence.

Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients.

Background & Aims: Somatic mutations provide uniquely specific markers for the early detection of neoplasia that can be detected in DNA purified from plasma or stool of patients with colorectal cancer. The primary purpose of the present investigation was to determine the parameters that were critical for detecting mutations using a quantitative assay. A secondary purpose was to compare the results of plasma and stool DNA testing using the same technology.

An electrophoretic capture method for efficient recovery of rare sequences from heterogeneous DNA.

It is difficult to isolate rare, PCR-quality DNA from specimens containing large quantities of nonspecific DNA from multiple sources (heterogeneous DNA). Extracting human DNA from stool for colorectal cancer (CRC) screening tests exemplifies this technically challenging sample preparation problem. The stool matrix is complex, the DNA composition heterogeneous, and CRC-associated mutated DNA is rare.

DNA integrity assay: a plasma-based screening tool for the detection of prostate cancer.

Purpose: The aim of this study was to evaluate the utility of the DNA integrity assay (DIA) as a plasma-based screening tool for the detection of prostate cancer.

Experimental Design: Blood samples were collected from patients with biopsy-proven prostate cancer prior to prostatectomy (n = 123) and processed as two-spin plasma preparations. The three control groups included: males <40 years old with no history of cancer (group 1, n = 20); cancer-free postprostatectomy patients (group 2, n = 25), and patients with a negative prostate biopsy (group 3, n = 22).

DNA stabilization is critical for maximizing performance of fecal DNA-based colorectal cancer tests.

We have developed a multitarget, fecal DNA screening assay that detects the presence of gene-specific mutations and long DNA fragments associated with colorectal cancer (CRC). We continue to investigate methods that may be used to optimize clinical sensitivity. The goals of this investigation are to establish how sample handling conditions affect the stability of DNA in stool, thereby potentially limiting clinical sensitivity, and to determine conditions to ameliorate DNA degradation.

Reproducibility of a multitarget stool-based DNA assay for colorectal cancer detection.

Objectives: Recent studies have demonstrated good sensitivity and specificity for the detection of colorectal cancer (CRC) utilizing a multitarget DNA assay panel (MTAP) on a single stool specimen. The aim of this study was to determine if analyzing three stool specimens obtained on three different days with the MTAP was superior to a single specimen for the detection of CRC. A secondary aim was to confirm the sensitivity of this MTAP reported in earlier studies.

DNA integrity as a potential marker for stool-based detection of colorectal cancer.

Background: Molecular genetic analysis of DNA in patient stools has been proposed for screening of colorectal cancer (CRC). Because nonapoptotic cells shed from tumors may contain DNA that is less degraded than DNA fragments from healthy colonic mucosa, our aim was to show that DNA fragments isolated from stools of patients with CRC had higher integrity than DNA isolated from stools of patients with healthy colonic mucosa.

Methods: We purified DNA from the stools of a colonoscopy-negative control group and patients with CRC and examined the relationship between long DNA fragments and clinical status by determining stool DNA integrity, using oligonucleotide-based hybrid captures with specific target sequences in increasingly long PCR reactions (200 bp, 400 bp, 800 bp, 1.

Sensitivity and specificity of a stool DNA multitarget assay panel for the detection of advanced colorectal neoplasia.

Colorectal cancer is the second-leading cause of cancer death. New noninvasive options for screening capable of diagnosing cancer at an early stage are needed to improve compliance and reduce mortality. This study was designed to provide an estimate of the sensitivity and specificity of a multitarget assay panel (MTAP) of stool DNA changes.

Accurate, noninvasive detection of Helicobacter pylori DNA from stool samples: potential usefulness for monitoring treatment.

A novel DNA assay demonstrating sensitive and accurate detection of Helicobacter pylori from stool samples is reported. Moreover, in three individuals tested for therapeutic response, the assay showed the disappearance of H. pylori DNA during treatment.

Stool screening for colorectal cancer: evolution from occult blood to molecular markers.

Background: Colorectal cancer is the second leading cause of malignant death, and better preventive strategies are needed. Participation rates for colorectal cancer screening remain low due, in part, to perceived discomfort, potential harm, and high costs with available tools.

Methods: Stool testing, unlike other conventional screening approaches, is noninvasive and requires no cathartic preparation.