Disseminated tumor cells in primary breast cancer: Evaluation of the percentage of breast cancer stem cells in bone marrow aspirates of patients receiving neoadjuvant chemotherapy

Abstract

505 Background: Disseminated tumor cells (DTC) in the bone marrow (BM) are related to poor prognosis of primary breast cancer (PBC). Cancer-initiating stem cells (CSC) have been detected in BM of PBC patients. Downregulation of the Notch pathway leads to murine mammary stem cell expansion and its expression is an early event in breast cancer. We enumerated CSC in BM mononuclear cells (BMC) and assessed the expression of Notch-1, ER, and HER-2 to study the differences between untreated patients and those treated with neoadjuvant chemotherapy. Methods: BM was collected from 90 PBC patients at time of surgery to assess DTC (CD326+CD45-) and CSC (CD326+CD45-CD44+CD24-) by FACS analysis. BM samples were also interrogated for the presence of CSC with ALDH activity using the Aldefluor® detection kit. RNA extracted from 61 BMC was analyzed for Notch-1, ER, and HER-2 transcripts by RT-PCR; the level of each transcript was normalized to that of the housekeeping gene, GAPDH. Results: The median age of patients is 51 years old (range, 27–77 years). A total of 29 patients (32%) received neoadjuvant chemotherapy, including 8 patients with anti-HER-2 targeted therapy. BMC of neoadjuvant-treated patients had a significantly higher median percentage of CSC in BMC (P= 0.046) and a significantly higher proportion of CSC cells within the Aldefluor+ population (P= 0.013) than those of untreated patients. Median level of Notch-1 transcripts in BMC was lower in neoadjuvant-treated patients compared with that of untreated patients (P= 0.07). There was an inverse correlation between the level of Notch-1 transcripts and the percentage of CSC in BMC of untreated (Spearman's rho= -0.519, P < 0.001), but not in neoadjuvant treated (Spearman's rho= -0.483, P= 0.058) patients. Conclusions: Neoadjuvant chemotherapy is associated with expansion of CSC in the BMC of patients with PBC. Untreated patients with higher level of Notch-1 transcripts in the BMC tend to have lower percentages of CSC and suggest that Notch signaling may be crucial for differentiation of CSC. The prognostic value of this finding is under investigation and gene expression profile of CSC may provide indications for novel neoadjuvant therapies. No significant financial relationships to disclose.