Abstract Purpose: Breast cancer cells with a CD44+CD24-/low phenotype have been suggested to have tumor-initiating properties with stem cell-like. The purpose of this study is to clarify the gene expression profiling of cells with different CD44/CD24 genotypes within breast cancer. Methods: Laser-captured microdissection was used to select the isolation of cancer cells in 36 frozen tissues of breast cancer, and RNA extracted from these cells was examined by real-time RT-PCR to quantify CD44 and CD24 expressions. Human stem cell RT2 profiler PCR array was used for gene expression analysis in the groups of different CD44/CD24 genotype. Associations between different CD44/CD24 genotypes and clinical variables were assessed by Fisher's exact test, and the Mann-Whitney U test was used for gene expression profiling. P<0.05 was considered significant. Results: Thirty-six tumors were divided into 3 groups. Group A was composed of CD44+CD24-/low genotype, in which the ratio of CD44/CD24 was >1.0. Group B was composed of CD44+CD24+ genotype, in which that was 0.1 < ratio ≤ 1.0. In group C of CD44-/lowCD24+ genotype, that was < 0.1. The number of tumors in group A, B and C was 5, 29 and 2, respectively. Regarding the correlation of CD44/CD24 status with tumor characteristics, tumors of group A were significantly associated with lymph node metastasis compared with those of group B (P=0.029). The number of tumor with HER 2 score 3 within group A and B was 0 and 11, respectively. There was no significantly difference in ER status and tumor size. As far as the signaling pathways, the number of expression genes for Notch pathway in group A was significantly greater than in group B (P=0.028), and that for Wnt pathway was not significantly different between group A and B. Overexpressed gene in group A was NUMB, which is related to the programmed cell death. MYST1 and SOX2 tended to show higher levels of group A than of group B. Conclusion: This study suggests that Notch pathway may be more important than Wnt pathway on breast cancer with CD44+CD24-/low genotype, and the CD44+CD24-/low status would be associated with low/negative HER2 expression. Moreover, the gene of self-renewal markers, such as MYST1, SOX2 and NUMB might be a target for therapy against breast cancer with low/negative HER2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4687.