Assessment of the tumor-immune microenvironment can be used as a prognostic tool for improved survival and as a predictive biomarker for treatment benefit, particularly from immune-modulating treatments including cytotoxic chemotherapy. Using digital spatial profiling (DSP), we studied the tumor-immune microenvironment of 522 breast cancer cases by quantifying 35 immune biomarkers on tissue microarrays from the MA.5 phase III clinical trial. In this trial, node-positive breast cancer patients were randomized to receive either non-anthracycline chemotherapy (cyclophosphamide, methotrexate, 5'-fluorouracil [CMF]) or anthracycline-containing cytotoxic chemotherapy (CEF). Donor block hematoxylin and eosin (H&E)-stained sections were scored for the level of stromal tumor-infiltrating lymphocytes (sTILs), according to the international guidelines. We hypothesized that patients with higher levels of tumor-immune infiltration, assessed by either DSP or H&E staining, would benefit from CEF (relative to CMF) more than patients with lower immune infiltration. Unsupervised hierarchical clustering of digitally scored biomarkers revealed 2 patient clusters: immune infiltrated versus ignored. Following a prespecified statistical plan crafted to meet REMARK (REporting recommendations for tumor MARKer prognostic studies) guidelines, we found that the DSP-derived Immune Cluster assignment did not predict an improved 10-year relapse-free survival for patients receiving CEF compared with CMF. However, a secondary hypothesis revealed a significant predictive value for H&E sTILs assessed on full-faced sections for CEF benefit over CMF in the entire cohort and the human epidermal growth factor receptor 2-enriched subset. As exploratory analyses, supervised clustering of DSP-scored biomarkers suggested that low levels of T-cell immunoglobulin and mucin domain 3 TIM-3 and high levels of human leukocyte antigen HLA-DR and programmed cell death protein ligand PD-L-1 are associated with sensitivity to CEF. Although novel high-plex techniques provide a detailed insight into the tumor microenvironment, conventional H&E staining remains a powerful tool that can be applied to full-faced sections to assess the value of the immune microenvironment, particularly sTILs, in predicting benefits from immunogenic chemotherapies.