scholarly journals Automated PCR detection of BRAF mutations in colorectal adenocarcinoma: a diagnostic test accuracy study

2015 ◽  
Vol 69 (5) ◽  
pp. 398-402 ◽  
Author(s):  
Richard Colling ◽  
Lai Mun Wang ◽  
Elizabeth Soilleux
Keyword(s):  
Digital Pcr ◽  
Turnaround Time ◽  
Test Accuracy ◽  
Braf Mutations ◽  
System A ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Accuracy Study ◽  
Automated Platform ◽  
Formalin Fixed

BackgroundTesting for BRAF mutations in colorectal carcinoma (CRC) is important in the screening pathway for Lynch syndrome and is of prognostic value to guide management. This is a diagnostic accuracy study of the Idylla system, a novel and automated alternative PCR system.Methods100 consecutive formalin-fixed, paraffin-embedded CRC resection cases were tested for BRAF mutations using the Idylla automated platform and compared with standard (Cobas) PCR.ResultsThe sensitivity of the Idylla BRAF test was 100% and the specificity was 96%. Only one discordant Idylla positive/standard PCR negative result occurred and on Droplet Digital PCR demonstrated a mutation not identified by traditional PCR in this case.ConclusionThis study has validated the Idylla system for BRAF testing in CRC and demonstrated a possibly greater sensitivity, in addition to cost effectiveness and shorter turnaround time, when compared with standard PCR.

scholarly journals Droplet Digital PCR for Mutation Detection in Formalin-Fixed, Paraffin-Embedded Melanoma Tissues

2018 ◽  
Vol 20 (2) ◽  
pp. 240-252 ◽  
Author(s):  
Ashleigh C. McEvoy ◽  
Benjamin A. Wood ◽  
Nima M. Ardakani ◽  
Michelle R. Pereira ◽  
Robert Pearce ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Single-day HER2neu amplification assessment using chip-based digital PCR in formalin-fixed paraffin-embedded breast carcinoma tissue

2018 ◽  
Vol Volume 10 ◽  
pp. 121-129
Author(s):  
Parth Shah ◽  
Shiva Murarka ◽  
Anupam Joshi ◽  
Bhavana Mehta ◽  
Vipal Parmar ◽  
...  
Keyword(s):  
Breast Carcinoma ◽  
Digital Pcr ◽  
Carcinoma Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Prevalence of low-penetrance KRAS (codons 12/13 and 61) and BRAF mutations in metastatic colorectal carcinoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14147-e14147
Author(s):  
Federico Rojo ◽  
Trinidad Caldes ◽  
Sandra Zazo ◽  
Miguel de la Hoya ◽  
Cristina Carames ◽  
...  
Keyword(s):  
Wild Type ◽  
Braf Mutations ◽  
Kras Mutations ◽  
Activating Mutations ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Intracellular Signal ◽  
Polymerase Chain ◽  
Current Guidelines ◽  
Formalin Fixed

e14147 Background: In patients with metastatic colorectal cancer (mCRC), activating mutations within KRAS, which result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC have been significantly associated with lack of response to cetuximab or panitumumab therapy. Although current guidelines recommend testing for frequent KRAS codons 12/13 mutations, emerging data indicate that additional KRAS and BRAF mutations are also predictive of non-responsiveness to anti-EGFR antibodies in mCRC. This study is aimed to analyze the prevalence of low-penetrance KRAS and BRAF V600 mutations in caucasian mCRC population. Methods: A two-institution retrospective cohort of 1,238 consecutive KRAS wild type mCRC patients previously studied for 7 mutations in codons 12/13 (G12D, G12A, G12V, G12S, G12R, G12C and G13D) by the CE-IVD marked ARMS-scorpion real-time polymerase chain reaction PCR (Therascreen, Qiagen) was assayed by the diagnostic TaqMelt PCR assay cobas KRAS mutation and cobas BRAF V600 mutation tests (Roche), which are designed to detect 19 mutations in KRAS codons 12, 13 and 61 (including G12F, G13C, G13R, G13S, G13A, G13V, G13I, Q61H, Q61K, Q61R, Q61L, Q61E and Q61P) and BRAF V600 (V600E, V600K and V600D) mutations. An additional cohort of 146 KRAS mutated patients by ARMS-scorpion PCR was studied. DNA was obtained by cobas DNA preparation kit from one single 5µm formalin-fixed paraffin-embedded tissue section. Results: In all samples, sufficient DNA was obtained for KRAS and BRAF mutational studies. Among 1238 KRAS codons 12/13 wild-type patients by ARMS-scorpion PCR,166 (13.4%) showed KRAS mutations, 117 (9.5%) in codons 12/13, and 49 (4%) in codon 61. BRAF V600 mutations were detected in 9% cases. In ARMS-scorpion PCR KRAS mutated patients, mutations were confirmed by cobas in all cases. Conclusions: The cobas mutation tests are robust and reproducible assays that, 1) detects a higher incidence (13.4%) of mutations in codons 12, 13, and 61 of the KRAS gene in wild-type mCRC population, 2) a relevant rate of BRAF mutations is present in the same population, and 3) requires a very small amount of tissue.

Comparison of Five Different Assays for the Detection of BRAF Mutations in Formalin-Fixed Paraffin Embedded Tissues of Patients with Metastatic Melanoma

2017 ◽  
Vol 21 (2) ◽  
pp. 209-216 ◽  
Author(s):  
Claire Franczak ◽  
Julia Salleron ◽  
Cindy Dubois ◽  
Pierre Filhine-Trésarrieu ◽  
Agnès Leroux ◽  
...  
Keyword(s):  
Metastatic Melanoma ◽  
Braf Mutations ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

A Standardized Staining Protocol for L1CAM on Formalin-Fixed, Paraffin-Embedded Tissues Using Automated Platforms

2014 ◽  
Vol 29 (2) ◽  
pp. 180-183 ◽  
Author(s):  
Mina Fogel ◽  
Ayelet Harari ◽  
Elisabeth Müller-Holzner ◽  
Alain G. Zeimet ◽  
Gerhard Moldenhauer ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Staining Procedure ◽  
Type I ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Staining Protocol ◽  
Automated Platform ◽  
Endometrial Carcinomas ◽  
L1 Cell Adhesion Molecule ◽  
Formalin Fixed

The L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers and can serve as a biomarker for prognosis in most of these cancers (including type I endometrial carcinomas). Here we provide an optimized immunohistochemical staining procedure for a widely used automated platform (VENTANA™), which has recourse to commercially available primary antibody and detection reagents. In parallel, we optimized the staining on a semi-automated BioGenix (i6000) immunostainer. These protocols yield good stainings and should represent the basis for a reliable and standardized immunohistochemical detection of L1CAM in a variety of malignancies in different laboratories.

scholarly journals Accuracy and Clinical Relevance of Intra-Tumoral Fusobacterium nucleatum Detection in Formalin-Fixed Paraffin-Embedded (FFPE) Tissue by Droplet Digital PCR (ddPCR) in Colorectal Cancer

Diagnostics ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 114
Author(s):  
José Guilherme Datorre ◽  
Ana Carolina de Carvalho ◽  
Mariana Bisarro dos Reis ◽  
Monise dos Reis ◽  
Marcus Matsushita ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Relevance ◽  
Fusobacterium Nucleatum ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection Accuracy ◽  
Assay Optimization ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

The use of droplet digital PCR (ddPCR) to identify and quantify low-abundance targets is a significant advantage for accurately detecting potentially oncogenic bacteria. Fusobacterium nucleatum (Fn) is implicated in colorectal cancer (CRC) tumorigenesis and is becoming an important prognostic biomarker. We evaluated the detection accuracy and clinical relevance of Fn DNA by ddPCR in a molecularly characterized, formalin-fixed, paraffin-embedded (FFPE) CRC cohort previously analyzed by qPCR for Fn levels. Following a ddPCR assay optimization and an analytical evaluation, Fn DNA were measured in 139 CRC FFPE cases. The measures of accuracy for Fn status compared to the prior results generated by qPCR and the association with clinicopathological and molecular patients’ features were also evaluated. The ddPCR-based Fn assay was sensitive and specific to positive controls. Fn DNA were detected in 20.1% of cases and further classified as Fn-high and Fn-low/negative, according to the median amount of Fn DNA that were detected in all cases and associated with the patient’s worst prognosis. There was a low agreement between the Fn status determined by ddPCR and qPCR (Cohen’s Kappa = 0.210). Our findings show that ddPCR can detect and quantify Fn in FFPE tumor tissues and highlights its clinical relevance in Fn detection in a routine CRC setting.

scholarly journals Next Generation Digital PCR Measurement of Hepatitis B Virus Copy Number in Formalin-Fixed Paraffin-Embedded Hepatocellular Carcinoma Tissue

2015 ◽  
Vol 61 (1) ◽  
pp. 290-296 ◽  
Author(s):  
Jing-Tao Huang ◽  
Ying-Juan Liu ◽  
Jin Wang ◽  
Zhi-Gao Xu ◽  
Ying Yang ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Copy Number ◽  
Digital Pcr ◽  
Hbv Infection ◽  
Copy Numbers ◽  
Formalin Fixed Paraffin ◽  
B Virus ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Abstract BACKGROUND Hepatocellular carcinoma (HCC) is strongly associated with hepatitis B virus (HBV) infection. False-negative results are common in routine serological tests and quantitative real-time PCR because of HBV surface antigen (HBsAg) variation and low HBV copy number. Droplet digital PCR (ddPCR), a next generation digital PCR, is a novel, sensitive, and specific platform that can be used to improve HBV detection. METHODS A total of 131 HCC cases with different tumor stages and clinical features were initially classified with a serological test as HBsAg positive (n = 107) or negative (n = 24) for HBV infection. Next, DNA templates were prepared from the corresponding formalin-fixed paraffin-embedded (FFPE) tissues to determine HBV copy number by ddPCR. RESULTS HBV copy numbers, successfully determined for all clinical FFPE tissues (n = 131), ranged from 1.1 to 175.5 copies/μL according to ddPCR. The copy numbers of HBV were positively correlated with tumor-nodes-metastasis (P = 0.008) and Barcelona-Clinic Liver Cancer (P = 0.045) classification. Moreover, serum cholinesterase correlated with hepatitis B viral load (P = 0.006). CONCLUSIONS HBV infection is a key factor that influences tumorigenesis in HCC by regulating tumor occurrence and development. ddPCR improves the analytical sensitivity and specificity of measurements in nucleic acids at a single-molecule level and is suitable for HBV detection.

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