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Cancer Responsiveness Monitoring based on Resistance mutations in CTCs

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Mutation detection in metastatic breast cancer

The majority of breast cancer-related deaths occur as a consequence of metastasis. Characterisation of metastatic tumour tissue may provide better treatment guidance for patients with progressive disease.

Fundamental Research
Health

Approximately 25 % of breast cancer patients will develop incurable metastatic disease. During metastasis, cancer cells dissociate from the primary tumour site and enter the lymphatic and blood system to colonise distant organs. Given the difficulty in accessing metastatic sites, circulating tumour cells (CTCs) may serve as proxies for the metastatic tumour. Based on this idea, the EU-funded CAREMORE (Cancer responsiveness monitoring based on resistance mutations in CTCs) project proposed to molecularly characterise CTCs in blood by combining two methods. The first method entailed the multiplex in situ detection of four proteins in CTCs and the results were compared to the primary tumour. The methodology assessed the presence and activity status of the human epidermal growth factor receptor 2 (HER2) – known to be overexpressed in breast cancer, and two proteins – p4E-bp and pS6, found downstream in the signal transduction pathway of PIK3CA. The assay employed fluorescence detection using two antibodies targeting the same protein and a nuclear stain on single cell CTCs, thus achieving double recognition and consequently, a higher specificity. For CTC isolation and profiling, the consortium developed a novel method that entailed placing cells from a buffy coat on a glass slide and performing double immunostaining for cytokeratin and a receptor for the epithelial cell adhesion molecule (EpCAM). Following identification, individual CTCs underwent digital PCR to detect the different mutation variations of the PIK3CA gene. Initial calibration of the assays was performed using reference cell lines but ultimately the approach was applied for therapy guidance in patients with metastatic breast cancer. In particular, the consortium wished to determine the treatment benefit for patients with HER2-positive CTCs and a HER2-negative primary tumour. Collectively, the work by the CAREMORE project addressed one of the major challenges of oncology, namely personalised medicine. Adapting therapy to the characteristics of individual patients is expected to maximise treatment response and minimise toxicity.

Keywords

Breast cancer, metastasis, circulating tumour cells, CAREMORE, PIK3CA

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