Frequency of cytokeratin positive stroma cells in bone marrow of patients with primary breast cancer – Standardized immunocytology and RT-PCR

Background: Bone marrow (BM) samples from 2017 patients operated for primary breast cancer (stage T1–4, N0/N+, M0) at the University Hospital Heidelberg between 1999 und 2005 were analysed for disseminated tumor cells (DTC) on cytospin slides using cytokeratin (CK) specific antibodies and the APAAP and SA-AP detection systems.

Material and Methods: Cytospin-slides (mean 6) with 1×10⁶ BM-cells (area 240mm²) were stained with 5D3 or A45 antibodies and analysed by automated picture analysis with the ACIS II system (ChromaVision).

Results: Criteria for classification of disseminated tumor cells were in compliance with the publication of Fehm et al. (Cancer, Vol.107, 2006). Using these criteria only 26 (1.3%) from 2017 patients with primary breast carcinoma were scored DTC positive. However, small cells with an excentrically located small and clear nucleus were identified at a frequency of 56%. Identical cell types were present in 39 (45.3%) from 86 normal bone marrow donors. These cells were identified as plastic-adherent recycling stem cells positive for the markers MUC1, EpCAM, HER–2 neuregulin and various cytokeratin polypeptides analysed at culture passages 1–5 by immunocytology and RT-PCR.

Discussion: We have identified stroma cells as the dominating cytokeratin positive cell type in the bone marrow of healthy donors and breast cancer patients. Elimination of these cells either by size exclusion in picture analysis or by immunomagnetic separation results in a tumor cell detection rates of less than 2% in patients with primary breast cancer, stage T1–4, N0/N+,M0. We conclude that cytokeratin-based DTC analysis at a level of 2×10⁶ BM cells has no clinical relevance.

DTC - stroma cells