Clinical utility of a liquid biopsy diagnostic approach in lung cancer using amplicon-based next-generation sequencing in parallel with allele-specific PCR.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e21516-e21516
Author(s):  
Yukti Choudhury ◽  
Wai Min Phyo ◽  
Jing Shan Lim ◽  
Chaitanya Gupta ◽  
Yiliang Ho ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Liquid Biopsy ◽  
Diagnostic Yield ◽  
Egfr Mutations ◽  
Driver Mutations ◽  
Advanced Lung Cancer ◽  
Parallel Testing ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

e21516 Background: The utility of a liquid biopsy test for diagnosis and treatment is determined by its accuracy, molecular target coverage and timeliness to a clinically informative result. We describe our clinical experience of providing comprehensive next-generation sequencing (NGS) plasma testing for advanced lung cancer cases, including parallel testing with allele-specific PCR (AS-PCR) for rapid EGFR mutation detection. Methods: Plasma cell-free DNA (cfDNA) from advanced lung cancer patients (n = 374) underwent real-world testing with an amplicon-based NGS assay, in a CAP and ISO15189 accredited central laboratory. The assay covers 51 genes, including 8 guideline recommended biomarkers- EGFR, BRAF, MET, ALK, RET, ROS1, ERBB2 and KRAS. 168 cases (44.9%) were treatment-naïve (baseline) and 206 cases (55.1%) had received one or more lines of treatment. Parallel testing with AS-PCR for 10 specific EGFR mutations was done for 151 cases (90 baseline, 61 non-baseline). Concordance of EGFR mutation detection by the two methods, and the frequency of detection of clinically actionable (driver) mutations by NGS were assessed. Turnaround time (TAT) was calculated from sample receipt. Results: An overall concordance of 97.4% was observed for EGFR mutations between AS-PCR and NGS. When restricted to baseline cases, concordance was 100%. Among baseline cases which were concordant with AS-PCR for EGFR negativity (n = 63), driver mutations were identified in EGFR (n = 7 rarer mutations), KRAS, ERBB2, MET, NRAS, BRAF and ALK (n = 16 total) by NGS, providing an additional 37% diagnostic yield (23/63 cases). Among all baseline cases tested by NGS, a driver gene mutation was found in 64.28% of cases, including EGFR (36.9%) and KRAS mutations (11.9 %). Among non-baseline cases which were EGFR-negative by NGS and by AS-PCR, added diagnostic yield provided by NGS was 33.3% (10 of 30 cases), and included 16.7% rarer EGFR mutations. Overall, detection rate of ALK, RET, ROS1 fusions was 2.4% (n = 5, 3 and 1, respectively). Median TAT for EGFR results by AS-PCR was 1 day (range 1-2 days), while median TAT for NGS results was 10 days (range 4-13 days). Conclusions: We report excellent real-world performance of blood-based liquid biopsy testing for detecting recommended biomarkers in lung cancers, including an approach combining two orthogonal platforms for quick decision making. Clinically meaningful diagnostic yields can be obtained using a timely comprehensive NGS assay, either individually or in parallel with rapid AS-PCR testing.

scholarly journals Liquid biopsy in colon cancer: comparison of different circulating DNA extraction systems following absolute quantification of KRAS mutations using Intplex allele-specific PCR

Oncotarget ◽  
2017 ◽  
Vol 8 (49) ◽  
pp. 86253-86263 ◽  
Author(s):  
Vera Kloten ◽  
Nadine Rüchel ◽  
Nadina Ortiz Brüchle ◽  
Janina Gasthaus ◽  
Nils Freudenmacher ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Dna Extraction ◽  
Liquid Biopsy ◽  
Absolute Quantification ◽  
Circulating Dna ◽  
Kras Mutations ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Acoustic array biochip combined with allele-specific PCR for multiple cancer mutation analysis in tissue and liquid biopsy

2021 ◽  
Author(s):  
Nikoletta Naoumi ◽  
Kleita Michaelidou ◽  
George Papadakis ◽  
Agapi E. Simaiaki ◽  
Roman Fernandez ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Liquid Biopsy ◽  
Point Mutations ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Braf V600e ◽  
Analytical Sensitivity ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Regular screening of cancerous point mutations is of importance to cancer management and treatment selection. Although excellent techniques like next-generation sequencing and droplet digital PCR are available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%, i.e., detection of 1 mutant copy of genomic DNA in an excess of 104 wild type molecules, was demonstrated; moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma, colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR, demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%. The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid biopsy.

Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods

2017 ◽  
Vol 55 (12) ◽  
Author(s):  
Carlos Martínez-Carretero ◽  
Fernando Iguaz Pascual ◽  
Antonio Rus ◽  
Ivan Bernardo
Keyword(s):  
High Resolution ◽  
Sanger Sequencing ◽  
High Resolution Melting ◽  
Receptor Gene ◽  
Screening Method ◽  
Egfr Mutations ◽  
Specific Pcr ◽  
Two Samples ◽  
Allele Specific ◽  
Allele Specific Pcr

AbstractBackground:The discovery of mutations in the epidermal growth factor receptor gene (Methods:We developed a screening method by high resolution melting (HRM) to detectResults:Eight samples showed discordant results between both methods. Three samples with negative results by allele specific PCR and positive by HRM were confirmed by Sanger sequencing (p.S768I+p.V769L, T751_I759del and p.E709K+p.G719A; patients 1, 3 and 4, respectively). One sample with a negative result by HRM, and positive by allele specific PCR (p.T790M; patient 2), was confirmed by Sanger sequencing. Additionally, two positive samples for a deletion in exon 19 by allele-specific PCR, were negative by Sanger sequencing and HRM (patients 2 and 5) and finally, two samples were negative by allele-specific PCR and positive by HRM and Sanger sequencing due to synonymous variants in exon 21.Conclusions:HRM is a good method for mutational screening in

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

scholarly journals Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhengjie Chen ◽  
Dengguo Tang ◽  
Jixing Ni ◽  
Peng Li ◽  
Le Wang ◽  
...  
Keyword(s):  
Marker Assisted Selection ◽  
Inbred Lines ◽  
Average Density ◽  
Snp Markers ◽  
Data Sets ◽  
Rna Seq ◽  
Specific Pcr ◽  
Maize Inbred Lines ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

scholarly journals Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 982
Author(s):  
Zhiliang Xiao ◽  
Congcong Kong ◽  
Fengqing Han ◽  
Limei Yang ◽  
Mu Zhuang ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Brassica Oleracea ◽  
Rapid Identification ◽  
Inbred Lines ◽  
Hybrid Lethality ◽  
Specific Pcr ◽  
Allele Specific ◽  
User Friendly ◽  
Allele Specific Pcr ◽  
Kasp Marker

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

Assessment of the prevalence of the 985A>G MCAD mutation in the French-Canadian population using allele-specific PCR

2007 ◽  
Vol 71 (6) ◽  
pp. 569-575 ◽  
Author(s):  
S Giroux ◽  
A Dubé-Linteau ◽  
G Cardinal ◽  
Y Labelle ◽  
N Laflamme ◽  
...  
Keyword(s):  
French Canadian ◽  
Specific Pcr ◽  
Canadian Population ◽  
Allele Specific ◽  
Allele Specific Pcr

scholarly journals Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

2020 ◽  
Vol 21 (23) ◽  
pp. 9083
Author(s):  
Catherine Taylor ◽  
Simi Chacko ◽  
Michelle Davey ◽  
Jacynthe Lacroix ◽  
Alexander MacPherson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Nsclc Patient ◽  
Egfr Mutations ◽  
Single Nucleotide Variants ◽  
Cell Free Dna ◽  
Free Dna ◽  
Nsclc Patients ◽  
Peptide Affinity

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

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