Increase EGFR Mutations Detection Rate in Lung Adenocarcinoma by Real-Time PCR Screening Followed by Direct Sequencing

2015 ◽  
Vol 23 (6) ◽  
pp. 470
Keyword(s):  
Lung Adenocarcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Direct Sequencing ◽  
Egfr Mutations ◽  
Pcr Screening

Increase EGFR Mutations Detection Rate in Lung Adenocarcinoma by Real-Time PCR Screening Followed by Direct Sequencing

2015 ◽  
Vol 23 (5) ◽  
pp. 343-348 ◽  
Author(s):  
Tze-Kiong Er ◽  
Chin-Wen Lin ◽  
Ta-Chih Liu ◽  
Chih-Chieh Chen ◽  
Li-Hsuan Wang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Direct Sequencing ◽  
Egfr Mutations ◽  
Pcr Screening

EGFR mutation rate in liquid-based cytology compared with biopsies from primary and metastatic lung cancer using real-time PCR.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e19052-e19052
Author(s):  
Simonetta Buglioni ◽  
Valerio D'Alicandro ◽  
Elisa Melucci ◽  
Cristiana Ercolani ◽  
Paolo Visca ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Real Time ◽  
Molecular Analysis ◽  
Real Time Pcr ◽  
Direct Sequencing ◽  
Clinical Importance ◽  
Egfr Mutations ◽  
Tumor Biopsy ◽  
Direct Dna Sequencing ◽  
Liquid Based Cytology

e19052 Background: Specific EGFR tyrosine kinase inhibitors (TKIs) are recommended as first-line therapy in patients with non-small cell lung cancer (NSCLC) that have active EGFR mutations. Although cytology and tumor biopsy are a very common approaches to evaluate patient eligibility to TKIs, the limited number of tumor cells present in these samples may often constitute a bias for molecular analysis. In this context, the use of sensitive methods to detect EGFR mutations may be of pivotal clinical importance. The aim of this study was two-fold: 1) to determine the accuracy and sensitivity in detecting EGFR mutations between liquid-based cytology (LBC) and bioptic specimens, 2) to compare the performance of direct DNA sequencing and real-time PCR (qPCR) in two different series of cytologic and histologic primary and metastatic NSCLC. Methods: DNA wasextracted from 537 specimens of NSCLC patients including 422 biopsies and 115 ThinPrep (TP) collected samples respectively. EGFR mutations were analyzed by direct sequencing (exons 19 and 21) in 284 biopsies and 68 TP samples and by qPCR (exons 18, 19, 20, 21) in 138 biopsies and 47 LBC samples. Results: The overall specimen insufficiency rate between LBC and biopsies was significantly higher by direct sequencing compared to qPCR (biopsies: 11.6% vs 7.2%) and (LBC: 14.7% vs 2.3%). In the series of 309 valuable cases analyzed by direct sequencing (251 bioptic and 58 LBC), we found 34 (11%) EGFR mutations (11.2% biopsies and 10.3% LBC ). Of interest, a higher percentage of EGFR mutant cases (18.1%) was observed in the 170 valuable cases analyzed by qPCR (11.1% biopsies and 21.4% LBC). Conclusions: Our data indicate that TP collected specimens seem to be more reliable than formalin fixed biopsies for molecular analysis, probably due to the better quality of the extracted DNA. Furthermore, qPCR enabled detection of EGFR mutations in samples with low tumor content for which conventional Sanger sequencing was not informative.

scholarly journals P3.02b-027 Detection of EGFR Mutations in Plasma of Lung Adenocarcinoma Patients Using Real-Time PCR and Mass Spectrometry

2017 ◽  
Vol 12 (1) ◽  
pp. S1202-S1203
Author(s):  
Rossella Bruno ◽  
Elisabetta Macerola ◽  
Vincenzo Condello ◽  
Cristiana Lupi ◽  
Alessandro Ribechini ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Lung Adenocarcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Egfr Mutations

Identification of real-time PCR-negative EGFR mutations by direct sequencing test.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22118-e22118
Author(s):  
Tao-Yeuan Wang ◽  
Chi-Kuan Chen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Egfr Mutation ◽  
Detection Efficiency ◽  
Direct Sequencing ◽  
Egfr Mutations ◽  
Molecular Diagnostic ◽  
Molecular Testing ◽  
Mutation Site ◽  
Pcr Test

e22118 Background: High percentage of patients with non-small cell lung cancer (NSCLC) has EGFR activating mutations in Taiwan and responses to EGFR inhibitors such as Tarceva (1, 2, 3). Timely molecular diagnosis is critical and molecular testing should be performed before treatment for the individual patient. It is important to develop an effective molecular diagnostic method for accurately detecting mutations and preventing false positive results. Therefore, we used a universal genetic detecting method (FemtoPath) to increase sensitivity of gene mutation detection and improve the sensitivity limitation (0.1%). Our previous reports indicated that FemtoPath/direct sequencing test is more sensitive, accurate and inexpensive than Cobas KRAS real- time PCR mutation test. Methods: In this study, we compared two molecular testing methods for the detection of EGFR mutation; the direct sequencing and the real-time PCR. Results: FemtoPath EGFR direct sequencing test identified mutations in 57 of the 73 tumors (77.03%) and Cobas EGFR mutation test identifies mutations in 53 of the 73 tumors (72.97%). Six negative results obtained by Real-Time PCR test were re-confirmed to be positive by direct sequencing test. From the results above, FemtoPath/direct sequencing test not only accurately detected all of the mutations, but also identified mutations adjacent toexons 18, 19, 20 and 21 of the EGFR gene. In 52 consensus cases of EGFR mutation obtained from both methods, there were 10 major discrepancies including two S768, four Exon 19 deletion, one L858, five T790, and one exon 20 insertion in this experiment. Conclusions: FemtoPath/direct sequencing test has lower invalid rate than Real-Time PCR test while fewer sample amount was required with increased detection efficiency of unknown mutation site.

scholarly journals A two minute liquid based sample preparation for rapid SARS-CoV2 real-time PCR screening: a multicentre evaluation

2020 ◽  
pp. 104720
Author(s):  
Eric C.J. Claas ◽  
Pieter W. Smit ◽  
Mario J.A.W.M. van Bussel ◽  
Harold Verbakel ◽  
Mohammed Taouil ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Screening

scholarly journals Determination of EGFR gene somatic mutations in tissues and plasma of patients with advanced non-small cell lung cancer

2016 ◽  
Vol 62 (6) ◽  
pp. 638-644
Author(s):  
O.I. Brovkina ◽  
M.G. Gordiev ◽  
A.N. Toropovskiy ◽  
D.S. Khodyrev ◽  
R.F. Enikeev ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Real Time ◽  
Cell Lung Cancer ◽  
Real Time Pcr ◽  
Test System ◽  
Small Cell ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Egfr Gene

The presence of activating mutations in the EGFR gene influences cell proliferation, angiogenesis, and increases metastatic ability; it has a significant impact on the choice of medical therapy of non-small cell lung cancer (NSCLC). The use of targeted therapy with tyrosine kinase inhibitors requires performance of appropriate genetic tests. The aim of this study was to design a real-time PCR-based diagnostic kit for fast and cheap of EGFR mutations testing in paraffin blocks and plasma, and kit validation using samples from patients with NSCLC, and also comparative estimation of diagnostic features of real-time PCR with wild type blocking and digital PCR for mutation testing in blood plasma. The study included 156 patients with various types of adenocarcinoma differentiation. It was designed a simple and efficient real-time PCR-based method of detecting L858R activating mutation and del19 deletion in the EGFR gene for DNA isolated from paraffin blocks. Kit for EGFR mutations was validated using 411 samples of paraffin blocks. The proposed system showed high efficiency for DNA testing from paraffin blocks: a concordance with results of testing with therascreen® EGFR RGQ PCR Kit (`Qiagen`, Germany) was 100%. It has been shown the possibility of using this test system for the detection of mutations in plasma

O-091 Semen microbiota in patients with astenozoospermia and healthy controls: cluster analysis of real-time PCR data

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Panacheva ◽  
D Pochernikov ◽  
E Voroshilina
Keyword(s):  
Cluster Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Bacterial Load ◽  
Rrna Gene ◽  
Rt Pcr ◽  
Silhouette Index ◽  
Enterococcus Spp ◽  
Lactobacillus Spp

Abstract Study question What are the differences in the semen microbiota composition of patients with asthenozoospermia and normospermia according to cluster analysis of PCR data? Summary answer The detection rate of 4 stable semen microbiota clusters and the dominant bacteria groups varied in patients with asthenozoospermia and normospermia. What is known already Most of the research dedicated to analyzing normal and pathological semen microbiota is based on 16S rRNA gene specific Next generation sequencing (NGS). It has shown that microbiota is represented by polymicrobial communities (clusters) that consist of microorganisms from different genera and bacteria phyla. Despite it being highly informative, NGS has several weaknesses: complex sample preparation, difficult sample intake control, long analysis process, complicated results interpretation, high cost of equipment and reagents. These factors make it virtually impossible to use this approach in routine medical practice. Quantitative real-time PCR (RT-PCR) is far more suitable for this. Study design, size, duration Patients included in the study (n = 301) came to the “Garmonia” Medical Center (Yekaterinburg, Russia) either seeking preconception care or for infertility treatment. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 171) — asthenozoospermia, Group 2 (n = 130) — normospermia. Participants/materials, setting, methods Semen microbiota was analyzed using RT-PCR kit Androflor (DNA-Technology, Russia). Cluster analysis was performed for 201 samples with the total bacterial load (TBL) of at least 103 GE/ml (asthenozoospermia = 96, normospermia = 105). Cluster analysis was conducted using the k-means ++ algorithm, scikit-learn. The Silhouette index and the Davies–Bouldin index (DBI) were used to confirm the stability of clusters. Main results and the role of chance Both in the samples with normospermia and asthenozoospermia, four stable microbiota clusters were distinguished. Cluster I was characterized by the prevalence of obligate anaerobes, Lactobacillus spp. were prevalent in Cluster II, Gram-positive facultative anaerobes were prevalent in Cluster III, Enterobacteriaceae/Enterococcus spp. were prevalent in Cluster IV. Cluster I was detected the most often in both groups. However, in normospermia it was represented by various obligate anaerobes without pronounced quantitative predominance of any bacteria group. In samples with asthenozoospermia one of the bacteria groups were prevalent in Cluster I: Bacteroides spp./Porphyromonas spp./Prevotella spp., Peptostreptococcus spp./Parvimonas spp. or Eubacterium spp. In samples with asthenozoospermia Cluster II was characterized by the prevalence of Lactobacillus spp., while in samples with normospermia other bacteria groups were present along with lactobacilli, mainly obligate anaerobes. In samples with normospermia Corynebacterium spp. and Streptococcus spp., typical of normal microbiota of male UGT, were prevalent in Cluster III. In samples with asthenozoospermia Cluster III were characterized by the prevalence of Staphylococcus spp. In samples with asthenozoospermia Lactobacillus spp was present in Cluster IV along with Enterobacteriaceae/Enterococcus spp., which was not typical of the samples with normospermia. Limitations, reasons for caution Cluster analysis was not conducted for the samples with TBL lower than 103 GE/ml, since their results were incompatible with the data received for the negative control samples. Wider implications of the findings Further research could determine the detection rate of the described bacterial clusters in semen with other pathologies. Establishing the relationship between the characteristics of semen microbiota and infertility in men might allow the development of new algorithms for treating patients with reproductive disorders, depending on the composition of semen microbiota. Trial registration number not applicable

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