Notch-1 expression is not related to the percentage of CD326+ cells in bone marrow aspirates of patients with primary breast cancer.

Abstract

Abstract Abstract #4030 Background: The presence of epithelial cells (CD326+CD45dim) in the bone marrow (BM), i.e. disseminated tumor cells (DTC), are related to poor prognosis of primary breast cancer (PBC). Notch signaling pathway is involved in the development of the breast and aberrant activation of Notch signaling is an early event in breast cancer. Others have shown that breast cancer stem cells (BCSC) with the putative phenotype of CD44+CD24- can be identified within cell lines and primary tumor and have upregulated genes including Notch-1. However, it is unclear if Notch-1 is highly expressed by DTC and BCSC in BM of patients with PBC. We hypothesized that the Notch-1 expression is correlated with the level of BCSC within DTC. In this study, we enumerated BCSC in DTC-enriched populations in patients with PBC and assessed the expression of Notch-1 by RT-PCR.
 Methods and Patients: As part of an ongoing prospective study, from September 2006 to May 2008, 121 BM aspirates were collected from as many patients with PBC. Thirty-seven patients, median age of 52 years (range, 30-76 years), provided BM aspirates under anesthesia at time of surgery. At the time of BM collection, 9 patients had received neoadjuvant therapy. BM mononuclear cells (BMC) were enriched for DTC using anti-CD326 antibody-coated magnetic beads, and isolated using the AutoMACS-Pro cell separator system. DTC and BCSC were identified by multi-color FACS analysis. Thereafter, DTC-enriched cell preparations were analyzed for expression of ER, PR, HER-2, Notch-1, and Numb by RT-PCR; level of each transcript was normalized to a ratio to the house keeping gene GAPDH.
 Results: The mean ± SEM DTC in BMC was 1.05 % ± 0.09% and the majority was BCSC (79.7% ± 1.87%). The proportion of DTC-enriched fractions that expressed Notch-1, Numb, ER, PR, and HER-2 transcripts was 27/37 (73.0%), 33/37 (89.2%), 24/37 (64.9%), 15/37 (40.5%), and 20/37 (54.1%), respectively. The expression of Notch-1, Numb, ER, PR, and HER-2 transcripts did not correlate with the percentage of DTC. Contrary to our hypothesis, Notch-1 and Numb mRNA expression in DTC-enriched cells negatively correlated with the percentage of BCSC (Spearman's rho = -0.384, p= 0.027; and rho= -0.347, p= 0.048, respectively).
 Conclusions: Our results show no correlation between the level of Notch-1 expression and the percentage of BCSC in DTC-enriched populations in BM of patients with PBC. Since these studies were conducted in CD326-enriched populations, it underscores the necessity to further purify the CD44+CD24- cells and conduct studies in isolated single cells to definitively delineate the gene expression of BCSC from patients with PBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4030.