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. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5' and 3' termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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. Methods: We examined DNA purified from the stool of 25 patients with colorectal cancers before surgery. In 16 of these cases, plasma samples also were available. Mutations in stool or plasma were assessed with an improved version of the BEAMing technology. Results: Of the 25 stool DNA samples analyzed, 23 (92%) contained mutations that were present in the corresponding tumors from the same patients. In contrast, only 8 of the 16 (50%) plasma DNA samples analyzed had detectable levels of mutated DNA. We found that the DNA fragments containing mutations in both stool and plasma DNA typically were smaller than 150 bases in size. The sensitivity of the new method was superior to a widely used technique for detecting mutations, using single base extension and sequencing, when assessed on the same samples (92% vs 60%; P = .008, exact McNemar test). Conclusions: When assessed with sufficiently sensitive methods, mutant DNA fragments are detectable in the stool of more than 90% of colorectal cancer patients. DNA purified from stool provides a better template for mutation testing than plasma.
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. cTRACE (chimeric Targeting Rare Alleles with CRISPR-based Enrichment) also applies this method to use Cas9/sgRNA to target loci for the addition of universal adapters, however it has an additional selection for specific mutations through the use of an allele-specific primer. These three methods can produce multiplex PCR that significantly reduces the optimization required for every target. The methods are also not specific to any downstream analytical platform. We additionally will present a mutation specific enrichment technology that is non-amplification based and leaves the DNA in its native state: TRACE (Targeting Rare Alleles with CRISPR-based Enrichment). TRACE utilizes the Cas9/sgRNA complex to sterically protect the ends of targeted sequences from exonuclease activity which digests both the normal variant as well as any off-target sequences.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract 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. Urine samples were analyzed for the presence of mutant FGFR3 and quantified matrix metalloproteinase 2 and the hypermethylation of TWIST1 and NID2 . A patient's chance of having (positive predictive value PPV) or not having (negative predictive value NPV) cancer was determined by FGFR3 alone or by all 4 biomarkers , respectively. Results Cystoscopy/biopsy diagnosed 690 of 748 patients as negative and 58 as positive for bladder cancer. Of 21 patients identified by FGFR3 as highly likely to have cancer, 20 were also positive by cystoscopy/biopsy, resulting in a PPV of 95.2% (20 of 21), with specificity of 99.9% (689 of 690). The 4-marker combination identified 395 patients as having a low likelihood of cancer. Of these, 56.2% (388 of 690) also had negative biopsy/cystoscopy findings, resulting in an NPV of 98.2% (388 of 395). In total, 416 of the 748 patients with hematuria (55.6%) were identified with extremely high NPV and PPV to have or not have bladder cancer. Conclusion This multianalyte assay accurately stratified patients with high confidence into those who likely do or do not have bladder cancer. This test was developed to enhance and not to eliminate referrals for urologic evaluation.
Background & Aims: Assay of altered DNA exfoliated into stool represents an intriguing approach to screen for colorectal neoplasia, but multiple markers must be targeted because of genetic ...heterogeneity. We explored the feasibility of a stool assay panel of selected DNA alterations in discriminating subjects with colorectal neoplasia from those without. Methods: Freezer-archived stools were analyzed in blinded fashion from 22 patients with colorectal cancer, 11 with adenomas ≥1 cm, and 28 with endoscopically normal colons. After isolation of human DNA from stool by sequence-specific hybrid capture, assay targets included point mutations at any of 15 sites on K-ras, p53, and APC genes; Bat-26, a microsatellite instability marker; and highly amplifiable DNA. Results: Analyzable human DNA was recovered from all stools. Sensitivity was 91% (95% confidence interval, 71%–99%) for cancer and 82% (48%–98%) for adenomas ≥1 cm with a specificity of 93% (76%–99%). Excluding K-ras from the panel, sensitivities for cancer were unchanged but decreased slightly for adenomas to 73% (39%–94%), while specificity increased to 100% (88%–100%). Conclusions: Assay of altered DNA holds promise as a stool screening approach for colorectal neoplasia. Larger clinical investigations are indicated.
GASTROENTEROLOGY 2000;119:1219-1227
Background: Increased DNA methylation is an epigenetic alteration that is common in human cancers and is often associated with transcriptional silencing. Aberrantly methylated DNA has also been ...proposed as a potential tumor marker. However, genes such as vimentin, which are transcriptionally silent in normal epithelium, have not until now been considered as targets for cancer-associated aberrant methylation and for use as cancer markers. Methods: We applied methylation-specific polymerase chain reaction to the vimentin gene, which is transcriptionally silent in normal colonocytes, and compared methylation of vimentin exon 1 in cancer tissues and in fecal DNA from colon cancer patients versus control samples from healthy subjects. Results: Vimentin exon-1 sequences were unmethylated in 45 of 46 normal colon tissues. In contrast, vimentin exon-1 sequences were methylated in 83% (38 of 46) and 53% (57 of 107) of tumors from two independently collected groups of colon cancer patients. When evaluated as a marker for colon cancer detection in fecal DNA from another set of colon cancer patients, aberrant vimentin methylation was detected in fecal DNA from 43 of 94 patients, for a sensitivity of 46% (95% confidence interval CI = 35% to 56%). The sensitivity for detecting stage I and II cancers was 43% (26 of 60 case patients) (95% CI = 31% to 57%). Only 10% (20 of 198 case patients) of control fecal DNA samples from cancer-free individuals tested positive for vimentin methylation, for a specificity of 90% (95% CI = 85% to 94%). Conclusions: Aberrant methylation of exon-1 sequences within the nontranscribed vimentin gene is a novel molecular biomarker of colon cancer and can be successfully detected in fecal DNA to identify nearly half of individuals with colon cancer.
Colon cancer is the third leading cause of cancer-related death in the United States, affecting ∼147,000 people each year.
Most colon cancers arise from benign neoplasms and evolve into ...adenocarcinomas through a stepwise histologic progression sequence
that starts from adenomas or hyperplastic polyps/serrated adenomas. Genetic alterations and, more recently, epigenetic alterations
have been associated with specific steps in this polyp-adenocarcinoma sequence and likely drive the histologic progression
of colon cancer. Consequently, we have assessed in colon adenomas and hyperplastic polyps the methylation status of MGMT , CDKN2A , and MLH1 to determine the timing and frequency of these events in the polyp-carcinoma progression sequence and subsequently to analyze
the potential for these methylated genes to be molecular markers for adenomas and hyperplastic polyps. We have found that
methylated MGMT , CDKN2A , and MLH1 occur in 49%, 34%, and 7% of adenomas and in 5%, 10%, and 7% of hyperplastic polyps, respectively, and that they are more
common in histologically advanced adenomas. Furthermore, analysis of fecal DNA from persons who have undergone colonoscopic
exams revealed methylated CDKN2A , MGMT , and MLH1 in fecal DNA from 31%, 48%, and 0% of individuals with adenomas and from 16%, 27%, and 10% of individuals with no detectable
polyps, respectively. These results show that aberrant methylated genes can be detected frequently in sporadic colon polyps
and that they can be detected in fecal DNA. Notably, improvements in the specificity and sensitivity of the fecal DNA-based
assays will be needed to make them clinically useful diagnostic tests for polyps.
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. However, widely used fecal blood tests yield frequent false-negative and false-positive results that lower screening effectiveness and raise program costs. There is a compelling biological rationale to target DNA alterations exfoliated from neoplasms into stool, and multiple DNA markers would need to be assayed because of the genetic heterogeneity of colorectal neoplasia. Early clinical studies with this multi-target DNA-based stool assay approach suggest high sensitivity for both colorectal cancer and premalignant adenomatous polyps while maintaining high specificity.
Conclusions: This apparently accurate and user-friendly new approach holds promise to improve the effectiveness, efficiency, and appeal of colorectal cancer screening. Large-scale clinical studies are clearly warranted to corroborate the early results.
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.
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, 1.3 kb, 1.8 kb, 24 kb). DNA fragments obtained from CRC patients were compared with fragments obtained from colonoscopy-negative individuals for length and/or integrity.
DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group.
The presence of long DNA fragments in stool is associated with CRC and may be related to disease-associated differences in the regulation of proliferation and apoptosis. An assay of fecal DNA integrity may be a useful biomarker for the detection of CRC.
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. Here we describe a new urine-based assay that uses ultradeep sequencing technology to detect single mutant molecules of fibroblast growth factor receptor 3 (FGFR3) DNA that are indicative of bladder cancer. Detection of FGFR3 mutations in urine would provide clinicians with a noninvasive means of diagnosing early-stage bladder cancer. The single-molecule assay detects FGFR3 mutant DNA when present at as low as 0.02% of total urine DNA and results in 91% concordance with the frequency that FGFR3 mutations are detected in bladder cancer tumors, significantly improving diagnostic performance. To our knowledge, this is the first practical application of next-generation sequencing technology for noninvasive cancer diagnostics.
PDF
