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 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

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.

Sign in / Sign up

Export Citation Format

Share Document