The number of circulating tumour cells (CTCs) from the blood, or disseminated tumour cells (DTC) from the bone marrow, has strong prognostic power in either early stage (EBC) or metastatic (MBC) breast cancer patients, with molecular characterisation of these cells a new frontier. We have examined epithelial mesenchymal plasticity (EMP), a developmental program hijacked by carcinoma cells during metastasis, in (i) CTCs, and DTCs sampled from a prospective cohort of EBC patients at St Vincent’s Hospital Melbourne (SVHM) at diagnosis and during follow-up 3, 6, 12 and/or 24 months after surgery (n=29), (ii) CTCs and DTCs sampled from a cohort of MBC patients undergoing hip replacement for skeletal complications of malignancy at SVHM (n=10), and CTCs sampled from locally advanced breast cancer (LABC) and MBC patients from Royal Perth Hospital RPH (n=9).
CTCs and DTCs were isolated using in house anti-EpCAM and anti-EGFR immunobeads in parallel and/or sequentially, as these two markers are enriched on epithelial and mesenchymal cells respectively. Following RNA extraction, a 43 gene assay panel was assessed by quantitative RT-PCR, and the results compared with normal healthy volunteer controls (n=24 bloods and 10 bone marrows).
A significant background was observed in all control samples with values ranging from very high to negligible, making detection of signals arising from captured CTC or DTC difficult. Levels of EpCAM and other genes were higher than those measured in controls in some cases, indicating the presence of CTCs, and sometimes in longitudinal samples. The best evidence for CTCs was found in a HER2-amplified MBC patient, with ~105 fold higher levels of HER2 mRNA, and higher levels of several other genes. Integration of batched data with longitudinal data from EBC is ongoing, as is alignment with clinical history.
CTCs and DTCs are detectable by this qRT-PCR analysis in a subset of women with either EBC, LABC or MBC, but are relatively rare, and our ability to molecularly profile them is compromised by detection of significant levels of many of our genes in blood cells that have bound to the immunobeads during the isolation process. Some molecular markers appear more prominently, and with lower background values, and may be of use in characterising and monitoring the CTC and DTC compartment.
The EMPathy BCN gratefully acknowledges the support of the National Breast Cancer Foundation (CG-10-04; http://www.empathybcn.org.au).