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We hypothesized that cells present in normal tissue that bear cancer stem cell markers may represent a cancer cell of origin or a microenvironment primed for tumor development, and that their presence may correlate with the clinically defined subtypes of breast cancer that show increased tumorigenicity and stem cell features.    Methods  Normal tissues sampled at least 5 cm from primary tumors (normal adjacent tissue) were obtained from 61 chemotherapy-naive patients with breast cancer treated with mastectomy. Samples were stained simultaneously with immunofluorescence for CD44/CD49f/CD133/2 stem cell markers. We assessed the association between CD44 + CD49f + CD133/2 +  staining in normal adjacent tissue and breast cancer receptor subtype (defined by the expression of the estrogen (ER), progesterone (PR), or human epidermal growth factor-2 (Her2) receptors). We also examined the correlation between CD44 + CD49f + CD133/2 +  immunofluorescence and each of two previously published gene signatures, one derived from stem-cell enriched tissue and one from BRCA mutated tissue expected to have defective DNA repair.    Results  Patients with triple negative breast cancer (ER – /PR – /HER2 – ) expressed CD44 + CD49f + CD133/2 +  in 9 of 9 normal adjacent tissue samples compared with 7 of 52 ER +  and/or Her2 +  tumors ( P  < 0.001). Further, expression of CD44 + CD49f + CD133/2 +  by normal adjacent tissue correlated positively with a stem cell-derived tumorigenic signature ( P  <0.001) and inversely with a defective DNA-repair signature ( P  <0.001).    Conclusion  Normal cells bearing cancer stem cell markers are associated with the triple negative receptor subtype of breast cancer. This study suggests stem cell staining and gene expression signatures from normal breast tissues represent novel tissue-based risk biomarkers for triple negative breast cancer. Validation of these results in additional studies of normal tissue from cancer-free women could lay the foundation for future targeted triple negative breast cancer prevention strategies.

Cancer stem cell markers are enriched in normal tissue adjacent to triple negative breast cancer and inversely correlated with DNA repair deficiency