BACKGROUND: Not all breast cancer patients benefit from neoadjuvant or adjuvant 
therapy, resulting in considerable undertreatment or overtreatment. New insights 
into the role of tumor-infiltrating immune cells suggest that their composition, 
as well as their functionality, might serve as a biomarker to enable optimal 
patient selection for current systemic therapies and upcoming treatment options 
such as immunotherapy.
METHODS: We performed several complementary unbiased in silico analyses on gene 
expression profiles of 7270 unrelated tumor samples of nonmetastatic breast 
cancer patients with known clinical follow-up. CIBERSORT was used to estimate 
the fraction of 22 immune cell types to study their relations with pathological 
complete response (pCR), disease-free survival (DFS), and overall survival (OS). 
In addition, we used four previously reported immune gene signatures and a CD8+ 
T-cell exhaustion signature to assess their relationships with breast cancer 
outcome. Multivariable binary logistic regression and multivariable Cox 
regression were used to assess the association of immune cell-type fractions and 
immune signatures with pCR and DFS/OS, respectively.
RESULTS: Increased fraction of regulatory T-cells in human epidermal growth 
factor receptor 2 (HER2)-positive tumors was associated with a lower pCR rate 
(odds ratio [OR] = 0.15, 95% confidence interval [CI] = 0.03 to 0.69), as well 
as shorter DFS (hazard ratio [HR] = 3.13, 95% CI = 1.23 to 7.98) and OS 
(HR = 7.69, 95% CI = 3.43 to 17.23). A higher fraction of M0 macrophages in 
estrogen receptor (ER)-positive tumors was associated with worse DFS (HR = 1.66, 
95% CI = 1.18 to 2.33) and, in ER-positive/HER2-negative tumors, with worse OS 
(HR = 1.71, 95% CI = 1.12 to 2.61). Increased fractions of γδ T-cells in all 
breast cancer patients related to a higher pCR rate (OR = 1.55, 95% CI = 1.01 to 
2.38), prolonged DFS (HR = 0.68, 95% CI = 0.48 to 0.98), and, in HER2-positive 
tumors, with prolonged OS (HR = 0.27, 95% CI = 0.10 to 0.73). A higher fraction 
of activated mast cells was associated with worse DFS (HR = 5.85, 95% CI = 2.20 
to 15.54) and OS (HR = 5.33, 95% CI = 2.04 to 13.91) in HER2-positive tumors. 
The composition of relevant immune cell types frequently differed per breast 
cancer subtype. Furthermore, a high CD8+ T-cell exhaustion signature score was 
associated with shortened DFS in patients with ER-positive tumors regardless of 
HER2 status (HR = 1.80, 95% CI = 1.07 to 3.04).
CONCLUSIONS: The main hypothesis generated in our unbiased in silico approach is 
that a multitude of immune cells are related to treatment response and outcome 
in breast cancer.