Abstract Abstract #4031 Background
 The presence of epithelial cells in bone marrow (BM), disseminated tumor cells (DTC), that are CD326+CD45- is a negative independent prognostic factor in primary breast cancer (PBC). Cancer-initiating stem cells (CSC) are purported to be CD44+CD24-, express aldehyde dehydrogenase 1 (ALDH), and possess self-renewal potential. We examined the expression of CD44, CD24, and ALDH on BM aspirates of patients with PBC.
 Methods
 As part of an ongoing prospective study, from September 2006 to May 2008, BM aspirates were collected from 47 PBC patients, median age of 52 yrs (range: 30-76 yrs). At time of BM collection, 9 patients had received neoadjuvant therapy. BM mononuclear cells were analyzed for DTC and CSC phenotype and ALDH function (Aldefluor® assay kit) by 6-color FACS analysis and data correlated with primary lesion tumor size, tumor markers (ER and PR; over-expressed HER-2 by FISH), nodal status, and neoadjuvant chemotherapy.
 Results
 The mean (±SEM) percentages of DTC and ALDHbr cells were 1.07% ± 0.09% and 2.17% ± 0.21%, respectively. Within the DTC population, the mean proportion of CSC and ALDHbr cells were 79.6% ±1.69% and 43.8% ± 3.4%, respectively. There was a statistically significant positive correlation between size of the primary tumor and the percentages of DTC (rho =0.364, p = 0.016, n = 43), DTC/ALDHbr (rho = 0.533, p = 0.28, n = 17), and CSC within the DTC/ALDHbr population (rho = 0.790, p = 0.004, n = 11). Specifically, the BM from patients whose primary tumors were <2 cm (T1) had statistically significantly fewer DTC (0.81% vs. 1.26%, p = 0.013), DTC/ALDHbr cells (0.82% vs. 1.40%, p= 0.035), and CSC within the DTC/ALDHbr population (88% vs. 96%, p = 0.009) compared to tumors >2cm (T2). Addition of Aldeflour® to the immunophenotype analysis further enriched the proportion of CSC phenotype from 79.1% in the DTC population to 92.3% in the DTC/ALDHbr population (p = 0.047). Of particular clinical relevance, patients who received neoadjuvant therapy had a near statistically significantly higher percentage of DTC than untreated patients (adjuvant 0.99% vs. neoadjuvant 1.41 %, p = 0.066) without a significant decrease in the proportion of CSC within the DTC population (adjuvant 78.6% vs. neoadjuvant 84.6%, p = 0.312), suggesting CSC were not eliminated by neoadjuvant therapy.
 Conclusion
 Epithelial cells with cancer-initiating stem cell-like phenotypes can be identified in BM aspirates of patients with PBC. The DTC population contained a larger subset of CD44+CD24- cells that overlapped with a smaller subset of ALDHbr cells. The increased percentage of CSC within the ALDHbr population and persistence following therapy further suggests that the relevance of both phenotypes to the mechanism responsible for recurrent disease in breast cancer warrants further investigation and validation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4031.