Frithiof Henrik, Aaltonen Kristina, Rydén Lisa
Division of Oncology and Pathology.
Division of Surgery, Department of Clinical Sciences Lund, Lund University, Lund; Department of Surgery, Skåne University Hospital, Malmö, Sweden.
Onco Targets Ther. 2016 Nov 16;9:7095-7103. doi: 10.2147/OTT.S118502. eCollection 2016.
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.
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.
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.
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.
原癌基因（）的扩增在10% - 15%的原发性乳腺癌中出现，导致HER - 2受体激活，促进癌细胞生长。肿瘤分类在原发性肿瘤组织和转移活检中确定。然而，恶性细胞在疾病进展过程中倾向于改变其表型。循环肿瘤细胞（CTC）分析可作为重复活检的替代方法。美国食品药品监督管理局批准的CellSearch系统可测定HER - 2蛋白，但不能测定基因。本研究的目的是优化一种基于荧光原位杂交（FISH）的方法，以定量测定基于CellSearch分离后的乳腺癌CTC中的扩增情况，并在患者样本中验证该方法。
使用添加了人表皮生长因子受体2（HER - 2）阳性乳腺癌细胞系SKBr - 3和BT - 474的健康供体血液，以及相应的阴性对照（HER - 2阴性的MCF - 7细胞系），设计了一个体外CTC模型系统。在CellSearch系统中分离后，CTC样本进一步富集并固定在显微镜载玻片上。用细胞角蛋白和4',6 - 二脒基 - 2' - 苯基吲哚二盐酸盐进行免疫细胞化学染色，在荧光显微镜下鉴定CTC。通过应用HER2 IQFISH pharmDx检测法优化基于FISH的程序，以评估乳腺癌CTC中的扩增状态。
使用细胞系作为阳性和阴性对照，建立了一种在CellSearch分离后确定单个乳腺癌CTC中扩增存在的方法。该方法在乳腺癌患者的血液中得到验证，表明六名患者中有一名在转移性疾病治疗期间获得了CTC扩增。
在CellSearch分离并进一步富集后，可以确定CTC的扩增状态。在预测乳腺癌患者对HER - 2靶向免疫治疗的反应方面，FISH优于对状态的蛋白质评估。该检测方法有可能识别出状态发生变化且可能从治疗调整中获益的患者。
