scholarly journals Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Cristin Roma ◽  
Claudia Esposito ◽  
Anna Maria Rachiglio ◽  
Raffaella Pasquale ◽  
Alessia Iannaccone ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Response To Treatment ◽  
Egfr Mutations ◽  
Clinical Samples ◽  
Training Set ◽  
Similar Frequency ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Taqman Pcr ◽  
Validation Set

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct=37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

scholarly journals K-ras Point Mutation Detection in Lung Cancer: Comparison of Two Approaches to Somatic Mutation Detection Using ARMS Allele-specific Amplification

2000 ◽  
Vol 46 (12) ◽  
pp. 1929-1938 ◽  
Author(s):  
Simon J Clayton ◽  
Frank M Scott ◽  
Jill Walker ◽  
Kay Callaghan ◽  
Kemal Haque ◽  
...  
Keyword(s):  
Point Mutation ◽  
Mutation Detection ◽  
Lung Tumor ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Real Time Quantitative Pcr ◽  
Allele Specific ◽  
Specific Amplification ◽  
Tumor Dna

Abstract Background: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMSTM allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. Methods: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. Results: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04–100%. Conclusions: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

Rapid HLA-B27 screening with real-time TaqMan PCR: a clinical validation in the Dutch population

2011 ◽  
Vol 49 (12) ◽  
Author(s):  
Elianne A. Roelandse-Koop ◽  
Bert Buisman ◽  
Erik J. van Hannen ◽  
Anneke van der Zee ◽  
Wouter Kortlandt ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Human Leukocyte ◽  
Clinical Samples ◽  
Correct Result ◽  
Hla B27 ◽  
Dutch Population ◽  
Exon 2 ◽  
Allele Specific ◽  
Taqman Pcr

AbstractHuman leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis.We developed a real-time TaqMan PCR based on the Dominguez method with a β-Globin PCR as internal control.A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit (With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods.

scholarly journals 3424 Serial Biomarker Monitoring Predicts Long Term Outcomes in Acute Graft Versus Host Disease

2019 ◽  
Vol 3 (s1) ◽  
pp. 114-114
Author(s):  
Hrishikesh Krishna Srinagesh ◽  
Hrishikesh Krishna Srinagesh ◽  
Urvi Kapoor ◽  
Mina Aziz ◽  
Kaitlyn Ben-David ◽  
...  
Keyword(s):  
Regression Model ◽  
Clinical Response ◽  
Corticosteroid Therapy ◽  
Acute Gvhd ◽  
Accurate Determination ◽  
Immunosuppressive Treatment ◽  
Response To Treatment ◽  
Training Set ◽  
Validation Set

OBJECTIVES/SPECIFIC AIMS: The first aim of the study is to evaluate the accuracy of serum biomarkers of acute GVHD measured after four weeks of corticosteroid therapy to predict 6 month NRM. The second aim of this study is to compare the accuracy of the biomarker algorithm to that of clinical response to corticosteroids after four weeks. The third aim of the study is to develop a novel regression model that uses weekly biomarker measurements over the first month of corticosteroid therapy to predict 6 month NRM. METHODS/STUDY POPULATION:. Patients who received HCT at one of 22 IRB-approved centers and provided blood samples to the Mount Sinai Acute GVHD International Consortium (MAGIC) biorepository and developed GVHD between January 2008 to May 2018 are included in this study. Patients were divided by time into a training set (Jan 2008-Dec 2015, n=233) for model development and a validation set (Jan 2015-May 2018, n=357) to evaluate the predictive performance of the model. The later time of the validation set was chosen deliberately to model contemporaneous GVHD treatment practices. The size of each group was designed so that there would be roughly equal numbers of deaths in both groups. RESULTS/ANTICIPATED RESULTS:. Serum concentrations of GVHD biomarkers after one month of corticosteroid therapy were measured in the validation set, and the predicted probability of NRM ($\hat{\rm p}$) was computed according to the previously published algorithm: $\log[-\log(1 - \hat{\rm p})]=-11.263 + 1.844({\rm logST}2)+ 0.577({\rm logREG}3\alpha)$. The performance of the biomarker algorithm was evaluated by creating receiver operating characteristic (ROC) curves and calculating the area under the curve (AUC) in the validation set. The AUC of the biomarker algorithm was a significantly better predictor of 6 month NRM than clinical response to treatment after four weeks of corticosteroids (0.84 vs. 0.64, p<0.001), which is a clinically relevant improvement in accuracy. To evaluate serial biomarker monitoring, serum biomarker concentrations will be measured weekly at five time points from treatment initiation to one month after corticosteroid therapy. We will use these values in the training set to develop a regression model for 6 month NRM that accounts for repeated biomarker measurements. The performance of this model will be tested in the validation set and the accuracy of the serial biomarker measurements will be compared to the accuracy of measuring biomarkers at the single time point after four weeks of corticosteroid therapy. An AUC improvement of 0.05 would be considered clinically significant. DISCUSSION/SIGNIFICANCE OF IMPACT: Clinical response to treatment after four weeks has been the standard endpoint in GVHD interventional trials for decades. If biomarkers measured at the same time more accurately predict long term mortality, this study would provide the basis for a novel endpoint in GVHD trials and enable more accurate determination of effect size of experimental interventions. An accurate biomarker algorithm will prove useful in guiding immunosuppressive treatment decisions for patients with GVHD. Patients identified by the algorithm as low-risk may benefit from reduced-dose corticosteroid therapy, potentially reducing lethal opportunistic infections. Patients identified as high-risk will be candidates for more intensive immunosuppression or investigational therapies. This precision medicine approach tailors therapy to the individual patient’s biology.

scholarly journals Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Raffaela Barbano ◽  
Barbara Pasculli ◽  
Michelina Coco ◽  
Andrea Fontana ◽  
Massimiliano Copetti ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Fast Method ◽  
Allele Specific ◽  
Taqman Pcr ◽  
Melanoma Patients ◽  
Braf V600 Mutation

Allele-Specific Loss Of The Mir-15a/16-1 Cluster Correlates With ZAP70 Expression In CLL Patients With 13q Deletion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3753-3753
Author(s):  
Angelo Veronese ◽  
Felice Pepe ◽  
Jessica Chiacchia ◽  
Sara Pagotto ◽  
Serena Veschi ◽  
...  
Keyword(s):  
Clinical Course ◽  
Genomic Region ◽  
High Expression ◽  
Rna Expression ◽  
Training Set ◽  
Exact Test ◽  
Number Variation ◽  
Allele Specific ◽  
Validation Set ◽  
13Q Deletion

Abstract Chronic lymphocytic leukemia, the most common form of adult leukemia in the Western world, is characterized by a heterogeneous clinical course, with indolent or progressive forms that entail profoundly different approaches to treatment. The biomarkers useful to predict the clinical course of CLL encompass cytogenetic abnormalities, protein expression (ZAP70, CD38) and IGHV mutation status. Among the many molecular features characterizing biological and clinical aspects of CLL patients, deletion of 13q14 and down-regulation of its related microRNAs (miR-15a and miR-16-1) are the most frequent aberrations in CLL. The involvement of the miR-15a and mir-16-1 in CLL has been extensively described by in vivo and in vitro studies and the genomic region encompassing the miR cluster results frequently deleted, but the mechanisms that regulate the expression of these miRs in CLL are still poorly understood. Here we demonstrate that the G to A single nucleotide polymorphism (SNP) rs115069827 prevents the maturation of miR-15a by reducing the binding of the DROSHA complex to the pri-microRNA 15a/16-1. The study of this functional SNP allowed us to discover a novel mechanism of transcription of the miR-15a/16-1 cluster, independent of DLEU2, its host gene. We demonstrate that the genomic region immediately upstream of the miR-15a/16-1, including the SNP, acts as transcriptional activator. The miR-15a/16-1 cluster is transcribed by allele-specific mechanisms: transcription of one allele is driven by the RNA Polymerase II at the DLEU2 promoter, while the other allele is transcribed by a mechanism that involves occupancy of the newly identified transcription activator region by RNA Polymerase III. Interestingly, the latter mechanism is dominant within CLL cells with mono-allelic 13q14 deletion and high expression of ZAP70 (Fisher's exact test p:0.0128) (Table1). Moreover, in our patient cohorts (training set n=28, validation set n=19) there are CLL samples with cells carrying 13q14 deletions that did not encompass the miR-15a/16-1 locus. Almost all of these show high expression of ZAP70 (Fisher's exact test training set p:0.0055; validation set p:0.0374) and primiR regulation by RPIII, in the presence of both alleles of miR-15a/16-1. By CNV analysis of a more telomeric region (∼30 kb, U59 DLEU2 region), that encompasses the second DLEU2 promoter, we demonstrate that these CLL cells carry a mono-allelic deletion of this region, which entails loss of the primiR regulation by the DLEU2 promoter ( occupied by RPII).Table 1primiR 15a/16-1 transcritpion by RPII independent mechanism correlates with ZAP70 expression in 13q deleted CLL.SampleFISHCopy number variation (CNV)ZAP70IGVHRelative expression after α amanitin treatment (2µg/ml)primiR 15a/16-1RNA PolymeraseU59 DLEU1miR 15a/16-1primiR15a/16-1pretRNAtyrpreACTBpreGAPDHLLC3113qnd-M↓=↓IILLC42NK+/--M=↓↓IIILLC1813q+/--M↓=↓IILLC0713q+/--U↓==IILLC45NK+/--M↓=↓IILLC50NK+/--M==↓IIILLC52A13q+/--M=nd↓IIILLC6113q+/--M↓=↓IILLC8113q+/--M↓=↓IILLC4713q+/--M==↓IIILLC03ANK+/--M↓=ndIICLL4Tw13q+/--M↓=↓IICLL3TwNK+/-+M==↓IIILLC0412tri/13q+/-+/++U=↓↓IIILLC6413q+/-+U=↓↓IIILLC7013q+/++/++U==↓IIILLC3613q+/-+M=↓↓IIILLC80NK+/-+/++M==↓IIILLC17ANK+/-+/++nd=nd↓III Overall these data may clarify important genetic features of the major subgroup of CLL patients and highlight the concept that regulation of the expression of the miR-15a/16-1 is the critical event in CLL pathogenesis. Being ZAP70 high expression associated with poor prognosis in CLL, we show that the concomitant presence of different mechanisms of transcriptional regulation of miR-15a and miR-16-1 could have different impacts on the pathogenesis/progression of the CLL. nd: not determined, M: IGVH mutated, U: IGVH unmutated, CNV: Copy number variation, NK, normal karyotype; 13q, deletion of 13q chromosome region; 12tri, 12 trisomy; +/+, presence of both alleles; +/-, monoallelic deletion; -, ZAP70≤20%; +, ZAP70>20%; ↓, downregulation of RNA expression after α amanitin treatment; =, no change in the RNA expression after α amanitin treatment; II, RPII; III, RPIII. Fisher's test: RPIII vs ZAP70, p:0.0128; RPIII vs IGVH, p:0.576 Disclosures: No relevant conflicts of interest to declare.

Discriminating Malignancy in Thyroid Nodules: The Nomogram Versus the Kwak and ACR TI-RADS

2020 ◽  
Vol 163 (6) ◽  
pp. 1156-1165
Author(s):  
Juan Xiao ◽  
Qiang Xiao ◽  
Wei Cong ◽  
Ting Li ◽  
Shouluan Ding ◽  
...  
Keyword(s):  
Thyroid Nodules ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Diagnostic Study ◽  
Diagnostic Efficiency ◽  
Training Set ◽  
Multivariable Logistic Regression Model ◽  
Predictive Values ◽  
Validation Set ◽  
Sensitivity Specificity

Objective To develop an easy-to-use nomogram for discrimination of malignant thyroid nodules and to compare diagnostic efficiency with the Kwak and American College of Radiology (ACR) Thyroid Imaging, Reporting and Data System (TI-RADS). Study Design Retrospective diagnostic study. Setting The Second Hospital of Shandong University. Subjects and Methods From March 2017 to April 2019, 792 patients with 1940 thyroid nodules were included into the training set; from May 2019 to December 2019, 174 patients with 389 nodules were included into the validation set. Multivariable logistic regression model was used to develop a nomogram for discriminating malignant nodules. To compare the diagnostic performance of the nomogram with the Kwak and ACR TI-RADS, the area under the receiver operating characteristic curve, sensitivity, specificity, and positive and negative predictive values were calculated. Results The nomogram consisted of 7 factors: composition, orientation, echogenicity, border, margin, extrathyroidal extension, and calcification. In the training set, for all nodules, the area under the curve (AUC) for the nomogram was 0.844, which was higher than the Kwak TI-RADS (0.826, P = .008) and the ACR TI-RADS (0.810, P < .001). For the 822 nodules >1 cm, the AUC of the nomogram was 0.891, which was higher than the Kwak TI-RADS (0.852, P < .001) and the ACR TI-RADS (0.853, P < .001). In the validation set, the AUC of the nomogram was also higher than the Kwak and ACR TI-RADS ( P < .05), each in the whole series and separately for nodules >1 or ≤1 cm. Conclusions When compared with the Kwak and ACR TI-RADS, the nomogram had a better performance in discriminating malignant thyroid nodules.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals POS0859 DEEP PHENOTYPING OF DERMATOMYOSITIS BASED ON LIPID FERROPTOSIS-RELATED GENES BY MACHINE LEARNING

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 684.1-684
Author(s):  
J. Q. Zhang ◽  
S. X. Zhang ◽  
R. Zhao ◽  
J. Qiao ◽  
M. T. Qiu ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Expression Profiles ◽  
Inflammatory Myopathy ◽  
Shanxi Province ◽  
Survival Prediction ◽  
Training Set ◽  
Redox Biology ◽  
Score Model ◽  
Validation Set

Background:Dermatomyositis (DM) is an idiopathic inflammatory myopathy with heterogeneous clinical manifestation that raise challenges regarding diagnosis and therapy1. Ferroptosis is a newly discovered form of regulated cell death that is the nexus between metabolism, redox biology, and rheumatic immune diseases2. However, how ferroptosis maintains the balance of lymphocyte T cells and affect disease activity in DM is unclear.Objectives:To investigate an ferroptosis-related multiple gene expression signature for classification by assessing the global gene expression profile, and calculate the lymphocyte T cells status in the different subsets.Methods:Gene expression profiles of skeletal muscle from DM samples were acquired from GEO database. GSE143323 (30 patients and 20 HCs) was selected as the training set. The GSE3307 contained 21 DM patients and was selected as the validation set. The 60 ferroptosis genes were obtained from previous literature3. The intersection of the global gene and ferroptosis genes was considered the set of significant G-Ferroptosis genes for further analysis. The “NMF” (R-package) was applied as an unsupervised clustering method for sample classification by using G-Ferroptosis genes expression microarray data from the training datasets. An ferroptosis score model was constructed. The performance of the ferroptosis genes-based risk score model constructed by the DM training set was validated in the batch-1 and batch-2 DM sets. Normalized ferroptosis genes training data was used to compare the ssGSEA scores of gene sets between the high risk and low risk group. The statistical software package R (version 4.0.3) was used for all analyses. P value < 0.05 were considered statistically significant.Results:We selected 54 significant G-Ferroptosis genes for further analysis in training set. There were 2 distinct subtypes (high-ferroptosis-score groups and low-ferroptosis-score groups) identified in G-Ferroptosis genes cohort which were also identified in validation datasets (Fig.1A, C, D). Metallothionein 1G (MT1G) was a characteristic gene of low-ferroptosis-score group. The characteristic genes of high-ferroptosis-score group were acyl-CoA synthetase family member 2(ACSF2) and aconitase 1(ACO1) (Fig.1B). Patients in high-ferroptosis-score group had a lower level of Tregs compared with that of low-ferroptosis-score patients in both training and validation set (P <0.05, Fig.1E).Conclusion:The biological process of ferroptosis is associated with the lever of Tregs, suggesting the process of ferroptosis may be involved in the disease progression of DM. Identificating ferroptosis-related features for DM might provide a new idea for clinical treatment.References:[1]DeWane ME, Waldman R, Lu J. Dermatomyositis: Clinical features and pathogenesis. Journal of the American Academy of Dermatology 2020;82(2):267-81. doi: 10.1016/j.jaad.2019.06.1309 [published Online First: 2019/07/08].[2]Liang C, Zhang X, Yang M, et al. Recent Progress in Ferroptosis Inducers for Cancer Therapy. Advanced materials (Deerfield Beach, Fla) 2019;31(51):e1904197. doi: 10.1002/adma.201904197 [published Online First: 2019/10/09].[3]Liang JY, Wang DS, Lin HC, et al. A Novel Ferroptosis-related Gene Signature for Overall Survival Prediction in Patients with Hepatocellular Carcinoma. International journal of biological sciences 2020;16(13):2430-41. doi: 10.7150/ijbs.45050 [published Online First: 2020/08/08].Acknowledgements:This project was supported by National Science Foundation of China (82001740).Open Fund from the Key Laboratory of Cellular Physiology (Shanxi Medical University) (KLCP2019) and Innovation Plan for Postgraduate Education in Shanxi Province (2020BY078).Disclosure of Interests:None declared

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