Abstract P2-03-01: Identification of cancer stem cells (CD44+CD24−/lo) in bone marrow as a prognostic factor in early breast cancer patients
Authors: Giordano A , Gao H , Cohen EN , Anfossi S , Hess KR , Krishnamurthy S , Tin S , Cristofanilli M , Hortobagyi GN , Woodward WA , Ueno NT , Lucci A , Reuben JM 
Published: August 2014

Abstract
Background: Cancer stem cells (CSCs) in bone marrow (BM) are epithelial tumor cells that express CD44+CD24−/lo. CSCs can be further divided into those that have ALDH activity (Aldefluor+) and those that do not. We hypothesized that if CSCs are responsible for tumor dissemination, their presence in BM would be prognostic in early stages of breast cancer (EBC) patients. Therefore, using multiparameter flow cytometry (FACS), we assessed epithelial cells for expression of CD44+CD24−/low and Aldefluor in BM of EBC patients and correlated these findings with circulating tumor cells (CTC), disseminated tumor cells (DTC) and clinical outcome.
Methods: This is a prospective laboratory study (Protocol 04–0657) conducted in the Departments of Surgical Oncology and Hematopathology, at The University of Texas MD Anderson Cancer Center in Houston, TX, USA, and was approved by the institutional review board. BM aspirates were collected at the time of surgery from 108 patients with EBC. BM was analyzed for CSCs and ALDH activity by FACS analysis. Ten cytospin slides of the bone marrow mononuclear cells were prepared from each BM aspirate and reacted with a cocktail of antibodies, including AE1/AE3, CAM5.2, MNF116, CK8 and CK18, to identify epithelial cells (DTC) by immunohistochemistry (IHC). In addition, the CellSearch system (Veridex, Raritan, NJ) was used to enumerate CTCs in each of 3 tubes of 7.5 mL of peripheral blood per patient. Overall survival (OS) and disease-free survival (DFS) were calculated from the date of diagnosis and analyzed with Kaplan-Meier survival plots. Cox multivariate proportional hazards model was also performed.
Results: Of the 103 evaluable patients, 45 patients received neoadjuvant chemotherapy, 35 adjuvant chemotherapy and 23 no chemotherapy. The median follow-up was 40 months (range 7–66 months). Patients with CSCs in BM had a hazard ratio (HR) of 8.8 for DFS (p = 0.002); patients with Aldefluor+ CSCs had a HR of 5.9 (p = 0.052) for DFS. Seven patients have expired and all of them had CSCs in BM. Moreover, we evaluated the presence of DTCs by FACS (CD326+CD45−) in 104 patients. Sixty-one (75%) of patients had positive BM samples for DTC (CD326+CD45- BM cells ≥ 0.53%). No association was shown between DTC in BM as determined by FACS and CTCs in PB. In a multivariate model, after adjusting for age (&lt; 45 years old), clinical T stage, N stage, ER, PR, HER2 status, and nuclear grading, the presence of CSCs was found to be an independent predictor of DFS (HR = 15.8, P = 0.017) as was the presence of CTCs (HR = 13.9, P = 0.007). In this subset of patients, the presence of Aldefluor+ CSCs and DTCs was not found to be predictive of DFS in the multivariate model including the same factors as listed above.
Conclusions: Our data indicate that the presence of BM metastasis is correlated to CSCs, and CSCs irrespective of ALDH activity are independent adverse prognostic factors in EBC patients. Moreover, the presence of CTCs was a strong independent predictor of DFS.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-03-01.