e12123 Background: Chemotherapy for breast cancer destroys non–stem cells while sparing cancer stem cells (CSCs). In contrast, anti–HER2 therapy may eliminate resistant cells because HER2 may be a key driver of CSCs. CSC biomarkers have been found to be prognostic of poor outcome and predictive of resistance to therapy. However, there are no comprehensive studies of the impact of anti-HER2 therapies on CSC–related biomarkers. We conducted a prospective biomarker determination study of breast CSCs characterized by CD44v expression and increased aldehyde dehydrogenase 1 (ALDH1) enzymatic activity or expression. Methods: In a prospective trial (ClinicalTrials.gov: NCT01688609), 18 patients with operable primary HER2+ breast cancer (≥T2 excluding inflammatory, any N; median age of 54 yrs) were treated with preoperative anti–HER2 therapy following the NeoALTTO trial dual therapy arm regimen, with a goal of identifying novel predictive biomarkers for pCR. Proportions of tumor cells with CSC characteristics, defined as CD44v+ and ALDH1+, were estimated at baseline, at 6 weeks (after therapy with lapatinib/trastuzumab) and at 18 weeks (after therapy with lapatinib/trastuzumab and paclitaxel) to assess adaptive response. We determined changes in the quantity and characteristics of CSC–related biomarkers during preoperative therapy and correlated them to tumor response. Results: Out of 18 patients, 8 (44%) had a pCR; 5 of these 8 patients (62%) were positive for CD44v staining on tumor cells at baseline and none were positive on the 6–week biopsy. In contrast, 6 of the 10 patients without pCR exhibited persistent levels, or enrichment of CD44v proportion and expression at 6 and 18 weeks (p = 0.0128). ALDH1 expression and other biomarkers were not statistically significant predictors of pCR. Conclusions: Enrichment of CD44v+ tumor cells after double anti–HER2 therapy may predict poor response to dual anti–HER2 therapy with cytotoxic chemotherapy. A second biopsy after the start of preoperative therapy may reflect biological changes useful for the guidance and application of therapeutic strategies for patients with HER2+ breast cancer. Clinical trial information: NCT01688609.