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

Authors Frithiof H, Aaltonen K, Rydén L

Received 11 August 2016

Accepted for publication 23 September 2016

Published 16 November 2016 Volume 2016:9 Pages 7095—7103

DOI https://doi.org/10.2147/OTT.S118502

Checked for plagiarism Yes

Review by Single-blind

Peer reviewers approved by Dr Akshita Wason

Peer reviewer comments 3

Editor who approved publication: Dr William Cho


Henrik Frithiof,1 Kristina Aaltonen,1 Lisa Rydén2,3

1Division of Oncology and Pathology, 2Division of Surgery, Department of Clinical Sciences Lund, Lund University, Lund, 3Department of Surgery, Skåne University Hospital, Malmö, Sweden

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.

Keywords: neoplastic cells, circulating, breast neoplasms, human epidermal growth factor receptor 2, in situ hybridization, fluorescence, immunomagnetic separation

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