Background: Prediction of benefit from trastuzumab in patients (pts) with HER2+ breast cancer remains an important goal. We sought to investigate the predictive value of quantitative measurement of HER2, HER3, HER4, EGFR, ER and PTEN protein expression on the benefit of trastuzumab in the phase III HER2+ adjuvant N9831 study for pts randomized to chemotherapy alone (Arm A) or chemotherapy with sequential (Arm B) or concurrent trastuzumab (Arm C). Methods: For each marker, we evaluated quantitative expression, relationship with demographic data, and association with disease-free survival (DFS) of pts. Freshly cut tissue microarray slides with up to three-fold redundancy per specimen from the N9831 cohort were treated identically using the AQUA (Camp, et al; Nat Med 2002, JCO 2008) method of quantitative immunofluorescence for each marker. HER2 was tested with CB11 (mouse monoclonal, Biocare, Inc.) and preliminary results were available for 698 of nearly 1400 pt specimens to be tested. The minimum value per pt was used in statistical analysis. Specimens were classified with high versus low expression based on a median value cutpoint for each marker. Median follow-up was 7.0 yrs. Results: Quantitative HER2 was compared with centrally performed HER2 testing by IHC and FISH. Median quantitative HER2 via AQUA was 10,017 units for the HER2 IHC 3+ group (n=607) versus 1058, 831, and 970 for the HER2 IHC 2+ (n=68), 1+ (n=11), and 0 (n=11) groups, respectively. The Spearman correlation between quantitative HER2 and FISH HER2/CEP17 ratio was 0.32 (p Conclusions: Similar to results based on standard HER2 testing by IHC and FISH in N9831, quantitative HER2 did not impact benefit from adjuvant trastuzumab. Results for additional markers will be presented. Our complete quantitative results for a second epitope on HER2, HER3, HER4, ER and EGFR will be the first report of these markers in a large patient cohort in the adjuvant setting. Disease Free Survival Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD05-03.