Background: Gene expression profiling can be used to predict the likelihood of achieving a pathological complete response (pCR) to neoadjuvant chemotherapy (CT) in patients with primary breast cancer. We report a retrospective exploratory analysis to evaluate the utility of the PAM50 subtype assay in predicting pCR in women enrolled in the NeOAdjuvant Herceptin (NOAH) trial, which recruited 334 patients. Methods: Formalin-fixed paraffin-embedded core biopsies were collected from 156 patients randomized to receive doxorubicin/paclitaxel (AT) followed by CMF (Group 1; HER2-positive/negative), or the same regimen with trastuzumab (H) (AT+H→CMF+H; Group 2; HER2-positive). Total RNA was amplified, labeled, and hybridized onto Affymetrix 2.0 Plus microarrays. The PAM50 microarray-based assay was applied to 43 genes. The PAM50 Risk Of Relapse score based on subtype and Proliferation (RORP) was evaluated as a continuous variable and as low/medium/high-risk groups using previously reported cutoffs. pCR was defined by the absence of residual invasive tumor in the breast and axillary lymph nodes. Univariate logistic regression was used to evaluate the association of each signature with pCR. Results: Overall pCR rates were 21.5% (20/93) and 44.4% (28/63) in Groups 1 and 2, respectively (p<0.005). In Group 1, non-LumA tumors showed higher pCR rates than LumA tumors (27% vs. 0%, p<0.005). Low, medium and high RORP groups showed 0%, 19% and 37% pCR rates (p=0.02). In Group 2, all subtypes were identified and HER2-enriched tumors showed a non-significant increase in pCR rates compared to non-HER2-enriched tumors (53% vs. 34%). Low, medium and high RORP groups showed 17%, 36% and 75% pCR rates (p=0.01). In Group 2 the odds ratio for achieving a pCR in the high-risk RORP group was 15.0 compared to the low-risk group (p=0.029). Conclusions: HER2-positive tumors predicted to have a high risk of relapse by the PAM50 assay benefited substantially from H-based CT. Similar findings are observed in HER2-positive/negative tumors treated with CT only. The role of PAM50 and/or other genomics assays in predicting pCR and survival during treatment with anti-HER2 agents warrants further investigation.