Dataset Information

Validation of p53 Immunohistochemistry (PAb240 Clone) in Canine Tumors with Next-Generation Sequencing (NGS) Analysis.

ABSTRACT: In human medicine, p53 immunohistochemistry (IHC) is a common method that is used for the identification of tumors with TP53 mutations. In veterinary medicine, several studies have performed IHC for p53 in canine tumors, but it is not known how well it actually predicts the mutation. The aim of this study was to estimate the accuracy of the IHC method for p53 (clone PAb240) using a lab-developed NGS panel to analyze TP53 mutations in a subset of malignant tumors in dogs. A total of 176 tumors were analyzed with IHC and then 41 were subjected to NGS analysis; among them, 15 were IHC positive and 26 were negative, and 16 out of 41 (39%) were found to be inadequate for NGS analysis. Excluding the non-evaluable cases at NGS, of the remaining eight IHC-positive cases, six were mutants and two were wild-type. Among the 17 IHC-negative cases, 13 were wild type, and 4 were mutants. The sensitivity was 60%, specificity was 86.7%, and the accuracy was 76%. These results suggest that when using IHC for p53 with this specific antibody to predict mutation, up to 25% wrong predictions can be expected.

SUBMITTER: Brunetti B

PROVIDER: S-EPMC10000222 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Validation of p53 Immunohistochemistry (PAb240 Clone) in Canine Tumors with Next-Generation Sequencing (NGS) Analysis.

Brunetti Barbara B de Biase Dario D Dellapina Giulia G Muscatello Luisa Vera LV Ingravalle Francesco F Tura Giorgia G Bacci Barbara B

Animals : an open access journal from MDPI 20230301 5

In human medicine, p53 immunohistochemistry (IHC) is a common method that is used for the identification of tumors with <i>TP53</i> mutations. In veterinary medicine, several studies have performed IHC for p53 in canine tumors, but it is not known how well it actually predicts the mutation. The aim of this study was to estimate the accuracy of the IHC method for p53 (clone PAb240) using a lab-developed NGS panel to analyze <i>TP53</i> mutations in a subset of malignant tumors in dogs. A total of ...[more]

PMID: 36899756

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Project description:BACKGROUND:Next generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process. RESULTS:In this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data. CONCLUSIONS:We successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.

Dataset Information

Validation of p53 Immunohistochemistry (PAb240 Clone) in Canine Tumors with Next-Generation Sequencing (NGS) Analysis.

Publications

Validation of p53 Immunohistochemistry (PAb240 Clone) in Canine Tumors with Next-Generation Sequencing (NGS) Analysis.

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