Multiple biomarker expression on circulating tumor cells in comparison to tumor tissues from primary and metastatic sites in patients with locally advanced/inflammatory, and stage IV breast cancer, using a novel detection technology

Abstract

Patients with locally advanced/inflammatory breast cancer (LABC/IBC) face a high likelyhood of recurrence and prognosis for relapsed, or de novo stage IV metastatic breast cancer (MBC) remains poor. Estrogen (ER) and HER2 receptor expression on primary or MBC allow targeted therapies, but an estimated 10-18% of tumors do not exhibit these biomarkers and survival in these cases is even poorer. Variations in discordance rates for the expression of ER and HER2 receptors have been observed between primary and metastatic tumors and such discordances may lead to suboptimal treatment. Circulating tumor cells (CTCs) are considered the seeds of residual disease and distant metastases and their characterization could help guide treatment selection. To explore this possibility, we used multiple biomarker assessment of CTCs in comparison to primary and metastatic tumor sites. Thirty-six patients with LABC/IBC, or stage IV MBC were evaluated. Blood samples were procured prior to initiating or changing therapy. CTCs were identified based on presence of cytokeratin and nucleus staining, and the absence of CD45. A multimarker assay was developed to simultaneously quantify expression of HER2, ER, and ERCC1, a DNA excision repair protein. Novel fiber-optic array scanning technology (FAST) was used for sensitive location of CTCs. CTCs were detected in 82% of MBC and 62% LABC/IBC cases. Multiplex marker expression was successfully carried out in samples from18 patients with MBC and in 8 patients with LABC/IBC that contained CTCs. In MBC, we detected actionable discordance rates of 40 and 23%, respectively for ER and HER2 where a biomarker was negative in the primary or metastatic tumor and positive in the CTCs. In LABC/IBC, actionable discordances were 60 and 20% for ER and HER2, respectively. Pilot trials evaluating the effectiveness of treatment selections based on actionable discordances between biomarker expression patterns on CTCs and primary or metastatic tumor sites may allow for a prospective assessment of CTC-based individualized targeted therapies.

Fig. 1. Schematic of FAST technology: the laser stimulates fluorescence in labeled cells on a glass slide as it moves over a fiber-optic bundle. Sites of fluorescence are digitally captured

Fig. 2

Images of tumor cells labeled for biomarkers. Cell 1 (top row) is labeled for HER2, Cell 2 (middle row) is labeled for ER and cell 3 (bottom row) is labeled for ERCC1. The first column (a) is a composite image of CK (red), nucleus (blue) and biomarker (green). Subsequent columns show images of individually labeled targets: CK (b), biomarker (c) and nucleus (d) respectively

Fig. 3. Distribution of expression levels of moderate expressing cell lines along with background. The relative protein scores of 0, 1, 2, and 3 are shown