Relevance of Tumor-Infiltrating Immune Cell Composition and Functionality for Disease Outcome in Breast Cancer

Abstract

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.

Figure 1.

Overview of breast cancer subtypes based on inferred receptor status, intrinsic molecular subtype, and triple-negative breast cancer subgroup classification. TNBC subgroups are classified as defined by Lehmann et al. (29). The estrogen receptor (ER)–positive (n = 4906) and human epidermal growth factor receptor 2 (HER2)–positive (n = 1580) subtypes contain double cases, being the ER-positive/HER2-positive tumors (n = 812). ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; TNBC = triple-negative breast cancer.

Figure 2.

Distribution of immune cell–type fractions in breast cancer subtypes compared with healthy breast tissue. Fractions of each immune cell type were compared by means of two-sided Mann-Whitney U test for breast cancer subtypes based on inferred receptor status, intrinsic molecular subtypes, and triple-negative breast cancer subgroups as defined by Lehmann et al. (23). A green triangle indicates a higher immune cell–type fraction in breast cancer as compared with normal breast tissue. A purple triangle indicates a lower fraction in breast cancer as compared with normal breast tissue. The size of the triangle represents the area under the curve of the effect size of the shift in the distribution of immune cell–type fractions. ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; TNBC = triple-negative breast cancer.

Figure 3.

Bubble heat map for the predictive and prognostic values of immune cell–type fractions in breast cancer subtypes. Associations between fractions and (A) pathological complete response (pCR), (B) disease-free survival (DFS), and (C) overall survival (OS) were analyzed. A blue bubble indicates that a higher fraction is associated with lower pCR rate, shorter DFS, or shorter OS; a yellow bubble indicates that a higher fraction is associated with higher pCR rate, prolonged DFS, or prolonged OS. The size of the bubble indicates the statistical significance level. The predictive value of immune cell–type fractions in the neoadjuvant setting was assessed by multivariable binary logistic regression using pCR as outcome variable and age, T-stage, grade, lymph node involvement, estrogen receptor (ER) status, human epidermal growth factor receptor 2 (HER2) status, and treatment regimen as covariates. The prognostic value of immune cell–type fractions in the neoadjuvant and adjuvant settings was assessed by multivariable Cox regression analysis, with time to distant metastasis and time to death as outcome variables and age, tumor size, grade, lymph node involvement, ER status, HER2 status, and treatment regimen as covariates. DFS = disease-free survival; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; NK = natural killer; OS = overall survival; pCR = pathological complete response; TNBC = triple-negative breast cancer.

Figure 4.

Bubble heat map for the predictive and prognostic values of immune gene signatures in breast cancer subtypes. Associations between fractions and (A) pathologicaly complete response (pCR), (B) disease-free survival (DFS), and (C) overall survival (OS) were analyzed. Signatures identified by Desmedt et al. (14), Teschendorff et al. (15), Perez et al. (16), Gu-Trantien et al. (Tfh signature) (17), and a CD8+ T-cell exhaustion signature (18) were investigated. A blue bubble indicates that a higher fraction is associated with lower pCR rate, shorter DFS, or shorter OS; a yellow bubble indicates that a higher fraction is associated with higher pCR rate, prolonged DFS, or prolonged OS. The size of the bubble indicates the statistical significance level. DFS = disease-free survival; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; NK = natural killer; OS = overall survival, pCR = pathological complete response; TNBC = triple-negative breast cancer.