ERalpha-status of disseminated tumour cells in bone marrow of primary breast cancer patients

Abstract

Introduction: Isolated disseminated tumour cells (DTC) are regarded as surrogate markers for minimal residual disease in breast cancer. Characterisation of these cells could help understand the known limitations of adjuvant therapy. Of particular interest is their oestrogen-receptor (ER) status because endocrine adjuvant therapy remains a cornerstone of breast cancer treatment.

Methods: Bone marrow (BM) aspirates from 254 patients with primary breast cancer were included in this study. A double immunofluorescence staining procedure was established for the identification of cytokeratin (CK) positive/Eralpha-positive cells. ERalpha status of the primary tumour was assessed immunohistochemically using the same antibody against ERalpha.

Results: In 107 of 254 (42%) breast cancer patients, CK-positive cells could be detected in the BM. More than one DTC in the BM was observed in 38 of the 107 patients. The number of detected cells ranged between 1 and 55 cells per 2 x 10(6) mononuclear cells. DTCs demonstrated ERalpha positivity in 12% of the patients. The ERalpha expression was heterogeneous in 10 of the 38 (26%) patients with more than one DTC. The concordance rate of ERalpha status between primary tumour and DTC was 28%. Only 12 of 88 patients with ERalpha-positive tumours also had ERalpha-positive DTCs.

Conclusions: Primary tumours and DTCs displayed a concordant ERalpha status in only 28% of cases. Most of the DTCs were ERalpha negative despite the presence of an ERalpha-positive primary tumour. These findings further underline the distinct nature of DTCs and may explain the failure rates seen in conventional endocrine adjuvant therapy.

Figure 1

Oestogen receptor (ER) α staining of MCF-7 (positive control) and SKBR3 (negative control) breast cancer cells spiked in bone marrow. A: MCF-7 cancer cells as positive control for ERα-staining. B: SKBR3 cancer cells as negative control ERα-staining.

Figure 2

Typical cytomorphology (nuclear size clearly enlarged, high nuclear to cytoplasmic ratio) and immunophenotype (irregular cytoplasmic staining for cytokeratin, cytokeratin filaments can be seen) of a representative disseminated tumour cell from a breast cancer patient. The tumour cell is stained with an anti-cytokeratin-fluorescein isothiocyanate (green) (×40 oil immersion objective).

Figure 3

Immunophenotyping of disseminated tumour cells from patients with primary breast cancer. The tumour cells were stained with an anti-cytokeratin-fluorescein isothiocyanate (green) and anti-oestrogen receptor (ER)α detected by a secondary Alexa Fluor 594 labelled goat anti-rabbit antibody (red). Nuclei are stained blue with 4'6-diamidino-2-phenylindole (DAPI) (×40 oil immersion objective). A-F: Breast cancer patients with ERα-positive disseminated tumour cells. G-H: Clusters of ERα-positive disseminated tumour cells. I: Cluster of ERα-positive and ERα-negative tumour cells.