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 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 Droplet Digital PCR Measurement of HER2 Copy Number Alteration in Formalin-Fixed Paraffin-Embedded Breast Carcinoma Tissue

2013 ◽  
Vol 59 (6) ◽  
pp. 991-994 ◽  
Author(s):  
Phillip Belgrader ◽  
Stephanie C Tanner ◽  
John F Regan ◽  
Ryan Koehler ◽  
Benjamin J Hindson ◽  
...  
Keyword(s):  
Breast Carcinoma ◽  
Copy Number ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Invasive Breast Carcinoma ◽  
Chromosome 17 ◽  
Analysis Tool ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

BACKGROUND Human epidermal growth factor receptor 2 (HER2) testing is routinely performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) analyses for all new cases of invasive breast carcinoma. IHC is easier to perform, but analysis can be subjective and variable. FISH offers better diagnostic accuracy and added confidence, particularly when it is used to supplement weak IHC signals, but it is more labor intensive and costly than IHC. We examined the performance of droplet digital PCR (ddPCR) as a more precise and less subjective alternative for quantifying HER2 DNA amplification. METHODS Thirty-nine cases of invasive breast carcinoma containing ≥30% tumor were classified as positive or negative for HER2 by IHC, FISH, or both. DNA templates for these cases were prepared from formalin-fixed paraffin-embedded (FFPE) tissues to determine the HER2 copy number by ddPCR. ddPCR involved emulsifying hydrolysis probe–based PCR reaction mixtures containing the ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2] gene and chromosome 17 centromere assays into nanoliter-sized droplets for thermal cycling and analysis. RESULTS ddPCR distinguished, through differences in the level of HER2 amplification, the 10 HER2-positive samples from the 29 HER2-negative samples with 100% concordance to HER2 status obtained by FISH and IHC analysis. ddPCR results agreed with the FISH results for the 6 cases that were equivocal by IHC analyses, confirming 2 of these samples as positive for HER2 and the other 4 as negative. CONCLUSIONS ddPCR can be used as a molecular-analysis tool to precisely measure copy number alterations in FFPE samples of heterogeneous breast tumor tissue.

scholarly journals Multi-center real-world comparison of the fully automated Idylla™ microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer

2020 ◽  
Author(s):  
Ana Velasco ◽  
Fatma Tokat ◽  
Jesper Bonde ◽  
Nicola Trim ◽  
Elisabeth Bauer ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Real World ◽  
Diagnostic Testing ◽  
Molecular Methods ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
High Concordance ◽  
Formalin Fixed

Abstract Microsatellite instability (MSI) is present in 15–20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.

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