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A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer

SIMPLE SUMMARY: Patients with gastric cancer may present variations in the copy number of the HER2 gene in their primary tumors. The techniques used to detect these variations and HER2 overexpression render false positive and negative results with high frequency, and robust methodologies are require...

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Autores principales: Juarez, Ignacio, Toro-Fernandez, Juan Francisco, Vaquero-Yuste, Christian, Molina-Alejandre, Marta, Lasa, Inmaculada, Gomez, Remedios, Lopez, Adela, Martin-Villa, Jose Manuel, Gutierrez, Alberto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230392/
https://www.ncbi.nlm.nih.gov/pubmed/34200787
http://dx.doi.org/10.3390/biology10060516
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author Juarez, Ignacio
Toro-Fernandez, Juan Francisco
Vaquero-Yuste, Christian
Molina-Alejandre, Marta
Lasa, Inmaculada
Gomez, Remedios
Lopez, Adela
Martin-Villa, Jose Manuel
Gutierrez, Alberto
author_facet Juarez, Ignacio
Toro-Fernandez, Juan Francisco
Vaquero-Yuste, Christian
Molina-Alejandre, Marta
Lasa, Inmaculada
Gomez, Remedios
Lopez, Adela
Martin-Villa, Jose Manuel
Gutierrez, Alberto
author_sort Juarez, Ignacio
collection PubMed
description SIMPLE SUMMARY: Patients with gastric cancer may present variations in the copy number of the HER2 gene in their primary tumors. The techniques used to detect these variations and HER2 overexpression render false positive and negative results with high frequency, and robust methodologies are required to assess this amplification and confidently select patients who may benefit from HER2-specific monoclonal antibody-based therapies. We addressed this issue by molecular biology techniques using DNA samples from tumor or distal tissue of gastric cancer patients. The HER2 and a control (IFNG) gene were subjected to differential (diffPCR) and quantitative PCR (qPCR). A cut-off point above which patients can be deemed positive was set based on the HER2/IFNG ratio, achieved using DNA from 30 healthy donors. Both, diffPCR and qPCR, identified the presence of somatic HER2 amplifications in 25% of patients in DNA from tumoral tissue, but not distal, paired tissue samples. Immunohistochemistry and immunofluorescence detected HER2 overexpression in tumor, but not distal, tissue of the patients previously identified as HER2+ by diffPCR and qPCR. Thus, the molecular biology-based techniques herein reported can identify patients with HER2 gene amplification and suitable for immune-based therapies. ABSTRACT: We have applied two PCR techniques, differential PCR (diffPCR) and qPCR for the identification of HER2 gene amplifications in genomic DNA of tumor and distal gastric samples from patients with gastric cancer. The diffPCR technique consists of the simultaneous amplification of the HER2 gene and a housekeeping gene by conventional PCR and the densitometric analysis of the bands obtained. We established a cut-off point based on the mean and standard deviation analyzing the DNA of 30 gastric tissues from patients undergoing non-cancer gastrectomy. diffPCR and qPCR yielded consistent results. HER2-overexpression was detected in 25% of patients and was further confirmed by immunohistochemistry and immunofluorescence. The approaches herein described may serve as complementary and reliable methods to assess HER2 amplification.
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spelling pubmed-82303922021-06-26 A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer Juarez, Ignacio Toro-Fernandez, Juan Francisco Vaquero-Yuste, Christian Molina-Alejandre, Marta Lasa, Inmaculada Gomez, Remedios Lopez, Adela Martin-Villa, Jose Manuel Gutierrez, Alberto Biology (Basel) Article SIMPLE SUMMARY: Patients with gastric cancer may present variations in the copy number of the HER2 gene in their primary tumors. The techniques used to detect these variations and HER2 overexpression render false positive and negative results with high frequency, and robust methodologies are required to assess this amplification and confidently select patients who may benefit from HER2-specific monoclonal antibody-based therapies. We addressed this issue by molecular biology techniques using DNA samples from tumor or distal tissue of gastric cancer patients. The HER2 and a control (IFNG) gene were subjected to differential (diffPCR) and quantitative PCR (qPCR). A cut-off point above which patients can be deemed positive was set based on the HER2/IFNG ratio, achieved using DNA from 30 healthy donors. Both, diffPCR and qPCR, identified the presence of somatic HER2 amplifications in 25% of patients in DNA from tumoral tissue, but not distal, paired tissue samples. Immunohistochemistry and immunofluorescence detected HER2 overexpression in tumor, but not distal, tissue of the patients previously identified as HER2+ by diffPCR and qPCR. Thus, the molecular biology-based techniques herein reported can identify patients with HER2 gene amplification and suitable for immune-based therapies. ABSTRACT: We have applied two PCR techniques, differential PCR (diffPCR) and qPCR for the identification of HER2 gene amplifications in genomic DNA of tumor and distal gastric samples from patients with gastric cancer. The diffPCR technique consists of the simultaneous amplification of the HER2 gene and a housekeeping gene by conventional PCR and the densitometric analysis of the bands obtained. We established a cut-off point based on the mean and standard deviation analyzing the DNA of 30 gastric tissues from patients undergoing non-cancer gastrectomy. diffPCR and qPCR yielded consistent results. HER2-overexpression was detected in 25% of patients and was further confirmed by immunohistochemistry and immunofluorescence. The approaches herein described may serve as complementary and reliable methods to assess HER2 amplification. MDPI 2021-06-10 /pmc/articles/PMC8230392/ /pubmed/34200787 http://dx.doi.org/10.3390/biology10060516 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Juarez, Ignacio
Toro-Fernandez, Juan Francisco
Vaquero-Yuste, Christian
Molina-Alejandre, Marta
Lasa, Inmaculada
Gomez, Remedios
Lopez, Adela
Martin-Villa, Jose Manuel
Gutierrez, Alberto
A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer
title A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer
title_full A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer
title_fullStr A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer
title_full_unstemmed A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer
title_short A Reliable and Standardizable Differential PCR and qPCR Methodology Assesses HER2 Gene Amplification in Gastric Cancer
title_sort reliable and standardizable differential pcr and qpcr methodology assesses her2 gene amplification in gastric cancer
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8230392/
https://www.ncbi.nlm.nih.gov/pubmed/34200787
http://dx.doi.org/10.3390/biology10060516
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