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

INTRODUCTION

Amplification of the () 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 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine 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 amplification status in breast cancer CTCs.

RESULTS

A method for defining the presence of 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 amplification during treatment against metastatic disease.

CONCLUSION

amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of 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 status who may benefit from treatment adjustments.