scholarly journals Methylation of SDC2/TFPI2 and Its Diagnostic Value in Colorectal Tumorous Lesions

2021 ◽  
Vol 8 ◽  
Author(s):  
Lianglu Zhang ◽  
Lanlan Dong ◽  
Changming Lu ◽  
Wenxian Huang ◽  
Cuiping Yang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Methylation Level ◽  
False Negative ◽  
High Specificity ◽  
Left Colon ◽  
Pcr Assay ◽  
Tissue Samples ◽  
Healthy Control ◽  
Auc Value ◽  
Control Samples

Background:SDC2 methylation is a feasible biomarker for colorectal cancer detection. Its specificity for colorectal cancer is higher than 90%, but the sensitivity is normally lower than 90%. This study aims to improve the sensitivity of SDC2 detection through finding a high positive target from the false-negative samples of SDC2 detection based on analysis of the bowel subsite difference in methylation.Methods: Hypermethylated TFPI2 was identified in SDC2 hypomethylated colorectal cancer samples retrieved from TCGA database with the methylation level lower than 0.2. The methylation-specific PCR assay was developed and then evaluated using tissue samples (184 cancer and 54 healthy control samples) and stool samples (289 cancer, 190 adenoma, and 217 healthy control samples).Results:TFPI2 was hypermethylated in most SDC2 hypomethylated colorectal cancer samples. When the SDC2/TFPI2-combined PCR assay was performed in stool specimens, the AUC value of cancer vs. control was 0.98, with the specificity of 96.40% and sensitivity of 96.60%, and the AUC value of adenoma vs. control was 0.87, with the specificity of 95.70% and the sensitivity of 80.00%. The improvement in sensitivity was the most momentous in the left colon. As the detection index, the Ct value was better in improving the sensitivity of detection than the methylation level based on the 2−ΔΔCt value.Conclusion:TFPI2 can improve the sensitivity of SDC2 methylation–specific detection of colorectal tumorous lesions while maintaining high specificity, in particular reducing the missed detection of left colon cancer and adenoma.

scholarly journals Diagnostic value in colorectal tumorous lesions of SDC2/TFPI2 methylation based on bowel subsite difference

2021 ◽  
Author(s):  
Lianglu Zhang ◽  
Lanlan Dong ◽  
Changming Lu ◽  
Wenxian Huang ◽  
Cuiping Yang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Methylation Level ◽  
Precancerous Lesions ◽  
High Specificity ◽  
Diagnostic Value ◽  
Detection Sensitivity ◽  
Tissue Samples ◽  
Potential Biomarker ◽  
Healthy Control ◽  
Auc Value

AbstractBackgroundSDC2 methylation is a potential biomarker for colorectal cancer detection with specificity over 90%, but its sensitivity is usually less than 90%. The study aims to improve the sensitivity and specificity by adding TFPI2 methylation as a complement.MethodsTFPI2 was identified using colorectal cancer samples from TCGA database with SDC2 methylation level lower than 0.2. SDC2/TFPI2 methylation specific PCR was performed using tissue samples (184 cancer and 54 healthy control) and stool samples (289 cancer, 190 adenoma and 217 healthy control). Detection sensitivity was calculated among tumors from different biopsy locations.Results88% of 50 TCGA specimens of colorectal cancer with SDC2 methylation level lower than 0.2 showed TFPI2 methylation level higher than 0.2. SDC2/TFPI2 combined detection in stool specimens showed AUC value of 0.98 with the specificity of 96.40% and sensitivity of 96.60% for cancer vs control, AUC value of 0.87 with the specificity of 95.70% and sensitivity of 80.00% for adenoma vs control. The sensitivities were much higher than those of SDC2, and the improvement was most significant in lesions from left colon and rectum.ConclusionsThis research indicated that the addition of TFPI2 can reduce the miss detection rate of colorectal cancer and adenomas while maintaining high specificity, probably by finding neoplasms in left colon.The method is non-invasive and has good compliance, also avoiding the pain of bowel preparation and risk of cross infection during endoscopy, so it will provide an easy and precise tool for colorectal cancer and its precancerous lesions screening.

Detection of DNA stool mutations in colorectal cancer patients.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22074-e22074
Author(s):  
Nora Manoukian Forones ◽  
Jacqueline Miranda Lima ◽  
Yolanda Teixeira ◽  
Celia Aparecida Marques Pimenta ◽  
Helena Murray ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Sanger Sequencing ◽  
Tissue Sample ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Screening Tests ◽  
Common Mutation ◽  
Tissue Samples ◽  
Human Dna ◽  
Control Samples

e22074 Background: Colorectal cancer (CRC) is the third leading cause of cancer in the world. Noninvasive screening tests have higher compliance. Changes in tumoral cells are also found in the stools after cancer cell exfoliation. Aim: to detect mutations in the stools of CRC patients and compare to mutations found in CRC tissue Methods: Patients referred for colonoscopy were divided into 3 groups: control, cancer, and polyp. The DNA was extracted from tissue and stool and it was analysed by two microarrays, the KRAS/BRAF/PIK3CA Array and the RanplexCRC Array (Randox Laboratories Ltd). Both based on the simultaneous detection of 20 mutations in KRAS, BRAF and PIK3CA and 28 mutations in KRAS, BRAF, TP53 and APC respectively. Positive tissue sample mutations were confirmed by Sanger sequencing. Results: 90 stools and 112 tissue biopsies were studied. Analysis of tissue samples was initially performed using the KRAS/BRAF/PIK3CA Array. In total mutations were detected in 16(55%) CRC, 12(45%) polyp and 2(6%) control samples. Mutations were confirmed via Sanger sequencing (76%). In the stool the frequency were 17(35%) in CRC, 10(71%) in polyp and 1(4%) in the control. Inconclusive tests were higher among the controls (32.5%) and polyps (22%), being less frequent in the CRC patients (6%). Excluding the inconclusive tests, WT genes were common in the controls (96%). The most common mutation was within KRAS codon 12. Stools samples from 48 subjects were compared to the results obtained by the RanplexCRC Array and mutations were found in 50% of CRC. KRAS/BRAF/PIK3CA Array stool results had a correlation with RanplexCRC Array in 64% of CRC patients and in 47% of polyp patients. The average human DNA in the stools was 15ng/ul in CRC patients and 0.46ng/ul in the controls (p=0.0001). Conclusions: Both arrays had a good correlation and a sensitivity between 30-50% with a high specificity (95-100%). The KRAS/BRAF/PIK3CA Array is currently optimized and recommended for assessment of tissue samples. This initial set of data however does indicate its potential applicability to stool specimens. Inconclusive tests are likely as a result of limited human DNA content within stool control samples. FAPESP (Sao Paulo Research Foundation) project 09/16618-8.

scholarly journals Immunohistochemical expression of β-catenin, Ki67, CD3 and CD18 in canine colorectal adenomas and adenocarcinomas

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kristin M. V. Herstad ◽  
Gjermund Gunnes ◽  
Runa Rørtveit ◽  
Øyvor Kolbjørnsen ◽  
Linh Tran ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
Colorectal Adenomas ◽  
Colorectal Carcinogenesis ◽  
Positive Staining ◽  
Colonic Tissue ◽  
Retrospective Case ◽  
Tissue Samples ◽  
Colonic Cells ◽  
Control Samples

Abstract Background Inflammation is believed to influence human colorectal carcinogenesis and may have an impact on prognosis and survival. The mucosal immunophenotype in dogs with colorectal cancer is poorly described. The aim of this study was to investigate whether the density, distribution and grade of tumor-infiltrating immune cells (TIIs) are different in normal colonic tissue vs benign stages (adenomas) and malignant stages (adenocarcinomas) of canine colorectal carcinogenesis, and thus, whether they can be considered as prognostic factors in dogs. This retrospective case-control study was performed on formalin-fixed, paraffin-embedded tissue samples from dogs with histologically confirmed colorectal adenoma (n = 18) and adenocarcinoma (n = 13) collected from archived samples. The samples had been collected by colonoscopy, surgery or during postmortem examination. Healthy colonic tissue obtained post mortem from dogs euthanized for reasons not involving the gastrointestinal tract served as control tissue (n = 9). Results The tumor samples had significantly lower numbers of CD3+ T-cells in the epithelium compared to controls (adenocarcinoma vs control, Kruskal-Wallis test, p = 0.0004, and adenoma vs control, p = 0.002). Adenomas had a significantly lower number of CD18+ cells in the lamina propria, compared to control samples (Kruskal-Wallis test, p = 0.008). Colonic samples from control dogs had uniform staining of β-catenin along the cell membrane of epithelial cells. Compared to normal colonic cells, the expression levels of cytoplasmic β-catenin were significantly higher in adenomas and adenocarcinomas (adenoma vs control Kruskal-Wallis test, p = 0.004, and adenocarcinoma vs control, p = 0.002). None of the control samples showed positive staining of β-catenin in the nucleus of colonic cells. In contrast, adenocarcinomas and adenomas showed moderate to strong staining of the cell nucleus. The nuclear β-catenin expression (signal strength and distribution) was significantly higher in adenomas compared to adenocarcinomas (Kruskal-Wallis test, p < 0.05). Conclusions β-catenin and Ki67 were not useful markers for demonstrating tumor progression from adenomas to adenocarcinomas. The lower presence of CD18 and CD3+ cells in colorectal tumors compared to controls indicates a reduced presence of histiocytes and T-cells, which may have implications for the pathogenesis and progression of colorectal cancer in dogs.

scholarly journals Meta-analysis of 16S rRNA Microbial Data Identified Distinctive and Predictive Microbiota Dysbiosis in Colorectal Carcinoma Adjacent Tissue

mSystems ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Zongchao Mo ◽  
Peide Huang ◽  
Chao Yang ◽  
Sihao Xiao ◽  
Guojia Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Random Forest ◽  
Colorectal Carcinoma ◽  
Large Scale ◽  
Colonic Cancer ◽  
Colorectal Carcinomas ◽  
Tissue Samples ◽  
Fecal Microbiome ◽  
Adjacent Tissue ◽  
Healthy Control

ABSTRACT As research focusing on the colorectal cancer fecal microbiome using shotgun sequencing continues, increasing evidence has supported correlations between colorectal carcinomas (CRCs) and fecal microbiome dysbiosis. However, large-scale on-site and off-site (surrounding adjacent) tissue microbiome characterization of CRC was underrepresented. Here, considering each taxon as a feature, we demonstrate a machine learning-based method to investigate tissue microbial differences among CRC, colorectal adenoma (CRA), and healthy control groups using 16S rRNA data sets retrieved from 15 studies. A total of 2,099 samples were included and analyzed in case-control comparisons. Multiple methods, including differential abundance analysis, random forest classification, cooccurrence network analysis, and Dirichlet multinomial mixture analysis, were conducted to investigate the microbial signatures. We showed that the dysbiosis of the off-site tissue of colonic cancer was distinctive and predictive. The AUCs (areas under the curve) were 80.7%, 96.0%, and 95.8% for CRC versus healthy control random forest models using stool, tissue, and adjacent tissue samples and 69.9%, 91.5%, and 89.5% for the corresponding CRA models, respectively. We also found that the microbiota ecologies of the surrounding adjacent tissues of CRC and CRA were similar to their on-site counterparts according to network analysis. Furthermore, based on the enterotyping of tissue samples, the cohort-specific microbial signature might be the crux in addressing classification generalization problems. Despite cohort heterogeneity, the dysbiosis of lesion-adjacent tissues might provide us with further perspectives in demonstrating the role of the microbiota in colorectal cancer tumorigenesis. IMPORTANCE Turbulent fecal and tissue microbiome dysbiosis of colorectal carcinoma and adenoma has been identified, and some taxa have been proven to be carcinogenic. However, the microbiomes of surrounding adjacent tissues of colonic cancerous tissues were seldom investigated uniformly on a large scale. Here, we characterize the microbiome signatures and dysbiosis of various colonic cancer sample groups. We found a high correlation between colorectal carcinoma adjacent tissue microbiomes and their on-site counterparts. We also discovered that the microbiome dysbiosis in adjacent tissues could discriminate colorectal carcinomas from healthy controls effectively. These results extend our knowledge on the microbial profile of colorectal cancer tissues and highlight microbiota dysbiosis in the surrounding tissues. They also suggest that microbial feature variations of cancerous lesion-adjacent tissues might help to reveal the microbial etiology of colonic cancer and could ultimately be applied for diagnostic and screening purposes.

scholarly journals DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening

2008 ◽  
Vol 54 (2) ◽  
pp. 414-423 ◽  
Author(s):  
Catherine Lofton-Day ◽  
Fabian Model ◽  
Theo DeVos ◽  
Reimo Tetzner ◽  
Juergen Distler ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Real Time ◽  
Restriction Enzyme ◽  
Real Time Pcr ◽  
Selection Process ◽  
Pcr Analysis ◽  
Plasma Samples ◽  
Tissue Samples ◽  
High Performing ◽  
Healthy Control

Abstract Background: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. Methods: We first used restriction enzyme–based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. Results: Restriction enzyme–based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%–73%] of plasma samples from CRC patients and not detected in 69% (62%–76%) of the controls. The corresponding results for NGFR were 51% (42%–60%) and 84% (77%–89%); for SEPT9, the values were 69% (60%–77%) and 86% (80%–91%). Conclusions: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

scholarly journals Evaluation of a panel of tumor-specific differentially-methylated DNA regions in IRF4, IKZF1 and BCAT1 for blood-based detection of colorectal cancer

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Graeme P. Young ◽  
Erin L. Symonds ◽  
Hans Jørgen Nielsen ◽  
Linnea Ferm ◽  
Ib J. Christensen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Sensitivity ◽  
High Specificity ◽  
Average Risk ◽  
Pcr Assay ◽  
Cross Sectional ◽  
Methylated Dna ◽  
Methylation Assay ◽  
Advanced Adenomas ◽  
Study Inclusion

Abstract Background Differentially-methylated regions (DMRs) are characteristic of colorectal cancer (CRC) and some occur more frequently than common mutations. This study aimed to evaluate the clinical utility of assaying circulating cell-free DNA for methylation in BCAT1, IKZF1 and IRF4 for detection of CRC. Methods A multiplexed real-time PCR assay targeting DMRs in each of the three genes was developed. Assay accuracy was explored in plasma specimens banked from observational cross-sectional trials or from volunteers scheduled for colonoscopy or prior to CRC surgery. Results 1620 specimens were suitable for study inclusion including 184 and 616 cases with CRC and adenomas, respectively, and 820 cases without neoplasia (overall median age, 63.0 years; 56% males). Combining the PCR signals for all targeted DMRs returned the best sensitivity for CRC (136/184, 73.9%, 95% CI 67.1–79.7), advanced adenomas (53/337, 15.7%, 95% CI 12.0–20.1) and high-grade dysplastic (HGD) adenomas (9/35, 25.7%, 95% CI 14.0–42.3) with a 90.1%, specificity for neoplasia (739/820, 95% CI 87.9–92.0, p < 0.01). Detection of methylation in all three genes were more likely in CRC cases than those without it (OR 28.5, 95% CI 7.3–121.2, p < 0.0001). Of the 81 positive cases without neoplasia, 62 (76.5%) were positive by a single PCR replicate only and predominantly due to detection of methylated BCAT1 (53.2%). Single replicate positivity was significantly higher than that in CRC (26/136, 19.1%, p < 0.0001), and single BCAT1 replicate positivity was more likely in cases without neoplasia than in CRC (OR 17.7, 95% CI 6.6–43.3, p < 0.0001). When a positive result was limited to those with ≥ 1 PCR replicate positive for either IKZF1 or IRF4, or at least two replicates positive for BCAT1, the multi-panel test maintained a high sensitivity for CRC (131/184, 71.2%, 95% CI 64.3–77.3) and HGD adenomas (8/35, 22.9%, 95% CI 11.8–39.3, p = 0.029) but improved specificity significantly (772/820, 94.1%, 95% CI 92.3–95.6, p < 0.0001 vs. any PCR replicate positive). Conclusion The multi-panel methylation assay differentiates cases with CRC from those without it and does so with high specificity when criteria for BCAT1 detection are applied. The marker panel is flexible and studies in those at average risk for CRC are now warranted to determine which panel configuration best suits screening goals. Trial registration: ACTRN12611000318987. Registered 25 March 2011, https://www.anzctr.org.au/ ACTRN12611000318987.

DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

2012 ◽  
pp. 15-19
Author(s):  
Thi Chau Anh Nguyen ◽  
Hoang Bach Nguyen ◽  
Hai Duong Huynh ◽  
Nu Xuan Thanh Le ◽  
Xuan Cuong Le ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rdna ◽  
False Negative ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Pcr Assay ◽  
Complex Objects ◽  
Method Performance ◽  
Gene Coding ◽  
16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

Scoring System for the Diagnosis of COVID-19

2020 ◽  
Vol 13 (1) ◽  
pp. 413-414 ◽  
Author(s):  
Mohamed Farouk Allam
Keyword(s):  
Scoring System ◽  
Clinical Symptoms ◽  
False Negative ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Negative Results ◽  
False Negative Results ◽  
Symptoms And Signs

Due to the international spread of COVID-19, the difficulty of collecting nasopharyngeal swab specimen from all suspected patients, the costs of RT-PCR and CT, and the false negative results of RT-PCR assay in 41% of COVID-19 patients, a scoring system is needed to classify the suspected patients in order to determine the need for follow-up, home isolation, quarantine or the conduction of further investigations. A scoring system is proposed as a diagnostic tool for suspected patients. It includes Epidemiological Evidence of Exposure, Clinical Symptoms and Signs, and Investigations (if available). This scoring system is simple, could be calculated in a few minutes, and incorporates the main possible data/findings of any patient.

scholarly journals A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study

2016 ◽  
Vol 55 (2) ◽  
pp. 180-184 ◽  
Author(s):  
Solène Le Gal ◽  
Florence Robert-Gangneux ◽  
Yann Pépino ◽  
Sorya Belaz ◽  
Céline Damiani ◽  
...  
Keyword(s):  
Real Time ◽  
Multicenter Study ◽  
Real Time Pcr ◽  
False Negative ◽  
False Negative Result ◽  
Pcr Assay
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