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A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation

INTRODUCTION: Amplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend...

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Autores principales: Frithiof, Henrik, Aaltonen, Kristina, Rydén, Lisa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Dove Medical Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117892/
https://www.ncbi.nlm.nih.gov/pubmed/27895501
http://dx.doi.org/10.2147/OTT.S118502
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author Frithiof, Henrik
Aaltonen, Kristina
Rydén, Lisa
author_facet Frithiof, Henrik
Aaltonen, Kristina
Rydén, Lisa
author_sort Frithiof, Henrik
collection PubMed
description INTRODUCTION: Amplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples. METHODS: Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4′,6-diamidino-2′-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs. RESULTS: A method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease. CONCLUSION: HER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of HER-2 status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in HER-2 status who may benefit from treatment adjustments.
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spelling pubmed-51178922016-11-28 A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation Frithiof, Henrik Aaltonen, Kristina Rydén, Lisa Onco Targets Ther Methodology INTRODUCTION: Amplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples. METHODS: Using healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4′,6-diamidino-2′-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs. RESULTS: A method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease. CONCLUSION: HER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of HER-2 status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in HER-2 status who may benefit from treatment adjustments. Dove Medical Press 2016-11-16 /pmc/articles/PMC5117892/ /pubmed/27895501 http://dx.doi.org/10.2147/OTT.S118502 Text en © 2016 Frithiof et al. This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.
spellingShingle Methodology
Frithiof, Henrik
Aaltonen, Kristina
Rydén, Lisa
A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation
title A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation
title_full A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation
title_fullStr A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation
title_full_unstemmed A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation
title_short A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation
title_sort fish-based method for assessment of her-2 amplification status in breast cancer circulating tumor cells following cellsearch isolation
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117892/
https://www.ncbi.nlm.nih.gov/pubmed/27895501
http://dx.doi.org/10.2147/OTT.S118502
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