scholarly journals A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0244332
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael J. Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Precancerous Lesions ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Total Dna

Background Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics. Methods Nineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance. Results The data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7–8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3–97.5%) and 92.2% (95% CI, 94.7–100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6–99.7%) and 92% sensitivity (95% CI, 86.1–95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%–99.1%) and 62.5% sensitivity (95% CI, 35.8%–83.7%) with AUC of 0.79 for precancerous lesions cfDNA. Conclusions The XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

scholarly journals A Novel Xenonucleic Acid Mediated Molecular Clamping Technology for Early Colorectal Cancer Screening

2020 ◽  
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael J. Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Precancerous Lesions ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Total Dna

Abstract BackgroundColorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM for early colorectal cancer diagnostics. MethodsNineteen mutations in four genes APC, KRAS, BRAF and CTNNB1 associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA) mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and a ROC cure was applied to evaluate its performance.ResultsThe data showed that the assay analytical sensitivity is 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5ng total DNA input per test. This assay is highly reproducible with intra-assay CV <3% and inter-assay <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%) respectively with AUC being about 0.96; and 96% (95% CI, 77.6-99.7%) specificity and 92% (95% CI, 86.1-95.6%) sensitivity with AUC 0.94 for CRC FFPE; and 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC 0.79 for precancerous lesions cfDNA.ConclusionsXNA mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

scholarly journals A Novel Xenonucleic Acid Mediated Molecular Clamping Technology for Early Colorectal Cancer Screening

2020 ◽  
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael J. Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Precancerous Lesions ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Total Dna

ABSTRACTBackgroundColorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape ™ for early colorectal cancer diagnostics.MethodsNineteen mutations in four genes APC, KRAS, BRAF and CTNNB1 associated with early events in CRC pathogenesis are targeted in the ColoScape™ assay. Xenonucleic Acid (XNA) mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and a ROC cure was applied to evaluate its performance.ResultsThe data showed that the assay analytical sensitivity is 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5ng total DNA input per test. This assay is highly reproducible with intra-assay CV <3% and inter-assay <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%) respectively with AUC being about 0.96; and 96% (95% CI, 77.6-99.7%) specificity and 92% (95% CI, 86.1-95.6%) sensitivity with AUC 0.94 for CRC FFPE; and 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC 0.79 for precancerous lesions cfDNA.ConclusionsXNA mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

A novel xenonucleic acid mediated molecular clamping technology for early colorectal cancer diagnostics.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e16106-e16106
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael Joseph Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Performance ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Performance Results

e16106 Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called ColoScape for early colorectal cancer diagnostics. Methods: Nineteen mutations in four genes APC, KRAS, BRAF and CTNNB1 associated with early events in CRC pathogenesis are targeted in the ColoScape assay. Xenonucleic Acid (XNA) mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were assessed for clinical performance, and a ROC curve was applied to evaluate its performance. Results: The data showed that the assay analytical sensitivity is 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5ng total DNA input per test. This assay is highly reproducible with intra-assay CV < 3% and inter-assay CV < 5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with different stages of CRC. The preliminary assay clinical specificity and sensitivity for cfDNA were 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%) respectively with AUC being about 0.96; and 96% (95% CI, 77.6-99.7%) specificity and 92% (95% CI, 86.1-95.6%) sensitivity with AUC 0.94 for FFPE. Conclusions: XNA mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of CRC mutations in cfDNA or FFPE samples.

A novel XNA-based NGS panel for cancer diagnostics.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e16142-e16142
Author(s):  
Qiang Gan ◽  
Zhen Cui ◽  
Simin Tang ◽  
Michael Joseph Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Lung Cancer ◽  
Colon Cancer ◽  
Allelic Frequency ◽  
Cancer Diagnostics ◽  
Analytical Sensitivity ◽  
Library Preparation ◽  
Wild Type ◽  
Assay Sensitivity ◽  
Clinical Sensitivity

e16142 Background: Identification of genetic variants with low allelic frequency using NGS method is confounded by the complexity of the human genome sequence and by bias and errors that arise during library preparation, sequencing and analysis. To overcome this, we developed a novel NGS approach employing a modified backbone nucleic acid molecule, Xenonucleic Acid (XNA) to enrich the target mutant before the sequencing. XNA enables to efficiently and selectively suppress wild type targeted DNA amplification but amplify targeted mutant alleles. Methods: We developed this XNA-based NGS for detection of mutations in lung and colorectal cancer which includes 8 genes (KRAS, NRAS, EGFR, BRAF, PIK3CA, APC, CTNNB1 and TP53) and 19 hotspots. Results: With as low as 10 ng input DNA, low allelic frequency mutant analysis powered by the XNAs in the library preparation increased the sensitivity of detection dramatically. There were, on average, 32, 24, 25 and 18-fold enrichment in variant allele frequency (VAF) for samples with original 0.10%, 0.25%, 0.50% and 1.25% VAF mutants, respectively. The analytical specificity is about 92% and analytical sensitivity (LOD) can be down to 0.10% VAF with 1000-2000X on Illumina MiSeq. The reproducibility results were obtained for intra-assays, inter-assays and inter-operator assays, and the CVs of detected VAF% were investigated. These 19 actionable mutants were validated by testing cfDNA and FFPE. Preliminary clinical sensitivity for FFPE sample is about 100% for lung cancer and colorectal cancer samples respectively, comparing to without XNA NGS about 85.7% for lung cancer and 70% for colon cancer. For cfDNA sample its clinical sensitivity is about 100% for lung and colon cancer, but without XNA mediated enrichment NGS is only about 70% for lung cancer and undetectable for early colon cancer. Conclusions: The result demonstrated that XNA can selectively block amplification of wild type alleles and leads to enrich of mutants read, and significantly increases the assay sensitivity without the requirement for NGS deeper sequencing.

scholarly journals Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255914
Author(s):  
Monia Ardhaoui ◽  
Emna Ennaifer ◽  
Anna Christina De Matos Salim ◽  
Flávio Marcom Gomez ◽  
Thalja Laasili ◽  
...  
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Multiple Infection ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Hpv Genotyping ◽  
Genotyping Method ◽  
Specificity And Sensitivity ◽  
Hpv Types

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

scholarly journals Target Enrichment Enhances the Sensitivity of Sanger Sequencing for BRAF V600 Mutation Detection

2021 ◽  
Author(s):  
Qiang Qan ◽  
Andrew Fu ◽  
Fang Liu ◽  
Shuo Shen ◽  
Maidar Jamba ◽  
...  
Keyword(s):  
Braf Mutation ◽  
Sanger Sequencing ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Target Enrichment ◽  
Mutation Status ◽  
Highly Sensitive ◽  
Braf V600 Mutation ◽  
Limited Sensitivity

BRAF is a serine/threonine protein kinase whose mutations lead to unregulated cell growth and cause different types of cancers. Since V600E is a major BRAF mutation and V600E detection as a companion diagnostic test (CDx) is stipulated in the labeling of the BRAF V600 inhibitors. Traditional Sanger sequencing cannot accurately detect mutations lower than 15% variant allele frequency (VAF) due to its limited sensitivity. Here we applied our patented XNA molecular clamping technology to modify Sanger sequencing template preparation by enriching the mutation population. We found that the use of our mutation-enriched template enhanced the analytical sensitivity of Sanger sequencing to 0.04% VAF. The method is verified to detect V600E, V600K, and V600R mutants and is validated for the known BRAF mutation status in clinical samples. Our streamlined protocol can be used for easy validation of the highly sensitive target-enrichment method for detecting BRAF V600 mutations using Sanger sequencing in clinical labs. In addition to BRAF V600 mutations, this method can be extended to the detection of other clinically important actionable mutations for cancer diagnostics.

COMPARATIVE EVALUATION OF MULTIPARAMETRIC ENDORECTAL ULTRASOUND AND ENHANCED IMAGING COLONOSCOPY IN THE DIAGNOSIS OF EARLY COLORECTAL CANCER

2020 ◽  
Vol 19 (3) ◽  
pp. 49-64
Author(s):  
E. M. Bogdanova ◽  
Yu. L. Trubacheva ◽  
O. M. Yugai ◽  
S. V. Chernyshov ◽  
E. G. Rybakov ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Statistical Significance ◽  
Muscle Layer ◽  
Endorectal Ultrasound ◽  
Diagnostic Methods ◽  
Early Colorectal Cancer ◽  
Morphological Examination ◽  
Optical Magnification ◽  
The Difference ◽  
Enhanced Imaging

AIM: to compare multiparametric endorectal ultrasound (ERUS) and enhanced imaging colonoscopy in the diagnosis of early colorectal cancer.PATIENTS AND METHODS: the study included 78 patients with epithelial rectal tumor. All the patients underwent multiparametric ERUS and colonoscopy with examination by narrow beam imaging (NBI) at optical magnification. All the patients were operated.RESULTS: a morphological examination removed specimens revealed adenomas in 48 cases, in 19 specimens – adenocarcinomas in situ and T1, and in 11 specimens – adenocarcinomas with invasion of the muscle layer or deeper. When calculating the accuracy indicators of diagnostic methods for groups of patients with adenoma, Tis-T1 adenocarcinoma, and T2-T3 adenocarcinoma, the difference in the sensitivity and specificity of the methods in none of the presented groups did not reach the level of statistical significance (p>0.05).ROC analysis showed that ultrasound has a prognostic value comparable to colonoscopy. The area difference was 0.013 (p=0.85).CONCLUSION: endoscopy and ultrasound have similar value in the diagnosis of malignant transformation of rectal adenomas.

Detectability of Plasma Proteins in SRM Measurements

2018 ◽  
Vol 16 (1) ◽  
pp. 74-81 ◽  
Author(s):  
Olga I. Kiseleva ◽  
Elena A. Ponomarenko ◽  
Yulia A. Romashova ◽  
Ekaterina V. Poverennaya ◽  
Andrey V. Lisitsa
Keyword(s):  
Blood Plasma ◽  
Human Plasma ◽  
Limit Of Detection ◽  
Human Blood Plasma ◽  
Analytical Sensitivity ◽  
Plasma Proteome ◽  
Protein Targets ◽  
Blood Plasma Sample ◽  
Specificity And Sensitivity ◽  
Human Plasma Proteome

Background: Liquid chromatography coupled with targeted mass spectrometry underwent rapid technical evolution during last years and has become widely used technology in clinical laboratories. It offers confident specificity and sensitivity superior to those of traditional immunoassays. However, due to controversial reports on reproducibility of SRM measurements, the prospects of clinical appliance of the method are worth discussing. </P><P> Objective: The study was aimed at assessment of capabilities of SRM to achieve a thorough assembly of the human plasma proteome. </P><P> Method: We examined set of 19 human blood plasma samples to measure 100 proteins, including FDA-approved biomarkers, via SRM-assay. </P><P> Results: Out of 100 target proteins 43 proteins were confidently detected in at least two blood plasma sample runs, 36 and 21 proteins were either not detected in any run or inconsistently detected, respectively. Empiric dependences on protein detectability were derived to predict the number of biological samples required to detect with certainty a diagnostically relevant quantum of the human plasma proteome. </P><P> Conclusion: The number of samples exponentially increases with an increase in the number of protein targets, while proportionally decreasing to the logarithm of the limit of detection. Analytical sensitivity and enormous proteome heterogeneity are major bottlenecks of the human proteome exploration.

Development, Popularization and Application of An Online OvAge Calculator: Qualitative Study Based on the WeChat Mini Program (Preprint)

2020 ◽  
Author(s):  
Wenwen Xu ◽  
Hui Wang ◽  
Zheng Zhu ◽  
Quan Wang ◽  
Jing Jin ◽  
...  
Keyword(s):  
Predictive Model ◽  
Ovarian Reserve ◽  
Chinese Women ◽  
Polycystic Ovary ◽  
Statistical Significance ◽  
Clinical Samples ◽  
Clinical Markers ◽  
Clinical Trial Registry ◽  
Specificity And Sensitivity ◽  
Short Period

BACKGROUND At present, there are many clinical markers and models to assess ovarian reserve, but none of them are ideal. The number of clinical samples is a key factor limiting the specificity and sensitivity of the markers and models, and traditional methods of subject recruitment are time and vigor. In addition, the model of ovarian reserve assessment for Chinese women need to be further explored. OBJECTIVE To explore the possibility of self-reporting for subjects through the WeChat mini program, and provide more data support for further optimization of the OvAge model, and to develop a predictive model of ovarian reserve that is specific to Chinese women. METHODS In this paper, with reference to the existing OvAge model, we developed an online OvAge calculator based on the WeChat mini program for data collection, and then applied the generalized linear model theory to obtain a predictive model of ovarian reserve which is in line with the characteristics of Chinese women. RESULTS Compared to traditional recruiting methods, the online OvAge calculator is able to collect a large number of samples in a short period of time, which is efficient and convenient. Optimized model of estimated OvAge =exp (3.5254-0.001*PRL-0.0231*AMH). This model showed a high statistical significance for each marker included in the equation. We applied the final equation on diminished ovarian reserve and polycystic ovary syndrome datasets and obtained a mean of predicted ovarian age significantly different from the mean of chronological age in both groups. CONCLUSIONS The OvAge calculator based on the WeChat mini program is a novel online subject self-reporting system that can collect many samples in a short period of time, continuously optimize the model and update the mini program version, which is economical, time-saving and efficient., and is worthy of promotion. In addition, the optimized OvAge model is suitable for Chinese women and provides a reference for clinical assessment of ovarian reserve. CLINICALTRIAL The study was approved by Chinese Clinical Trial Registry (registration No. ChiCTR2000037522) and Medical ethics committee of Jiangsu Province Hospital of Chinese Medicine (approved No. 2019NL-152-02).

Sign in / Sign up

Export Citation Format

Share Document