Tumor-infiltrating lymphocytes predict response to anthracycline-based chemotherapy in estrogen receptor-negative breast cancer

Abstract

Introduction: Infiltration of breast tumors by tumor-infiltrating lymphocytes (TIL) has been associated with sensitivity to anthracycline-based chemotherapy. However, it is unclear whether this is true within the estrogen receptor-alpha (ER)-negative subset of breast tumors that frequently manifest high TIL levels.

Methods: The association of TIL with short-term and long-term clinical response to anthracycline-based therapy was assessed in two independent ER-negative breast cancer cohorts in which patients were categorized as TIL-high or TIL-low. We defined an eight-gene lymphocyte mRNA expression signature (including CD19, CD3D, CD48, GZMB, LCK, MS4A1, PRF1, and SELL) and used unsupervised hierarchical clustering to examine the association between TIL and short-term response to neoadjuvant chemotherapy in a previously published cohort of ER-negative tumors (n = 113). We also examined the association between TIL and long-term chemotherapeutic efficacy in a second cohort of ER-negative tumors (n = 255) with longer than 6 years of median follow-up by using tissue microarrays and immunohistochemistry (IHC) for detection of CD3, CD8, CD4, CD20, and TIA-1.

Results: In patients with ER-negative tumors treated with neoadjuvant anthracycline-based chemotherapy, pathologic complete responses (pCRs) were achieved by 23 (74%) of 31 TIL-high patients and 25 (31%) of 80 TIL-low patients (odds ratio (OR), 6.33; 95% confidence interval (CI), 2.49 to 16.08; P < 0.0001). Multivariate logistic regression with standard clinicopathologic features demonstrated that only tumor size (P = 0.037) and TIL status (P = 0.001) were independent predictors of anthracycline response. In the second cohort, adjuvant anthracycline-based therapy was associated with increased disease-free survival (DFS) only in patients with high levels of intraepithelial CD3+ TIL (P = 0.0023). In contrast, outcomes after CMF treatment (cyclophosphamide, methotrexate, and fluorouracil) showed no association with CD3 status. In both cohorts, cytotoxic T-cells were the primary TIL subtype associated with anthracycline sensitivity. Finally, TIL significantly predicted anthracycline sensitivity for both the Her2-positive and triple-negative tumor phenotypes.

Conclusions: ER-negative breast cancers with high levels of TIL have heightened sensitivity to anthracycline-based chemotherapy, as assessed by the immediate response to neoadjuvant therapy and long-term outcome following adjuvant therapy. Investigations of TIL-based predictive tests to identify patients likely to benefit from anthracycline-based treatments are warranted.

Figure 1

An eight-gene tumor infiltrating lymphocyte (TIL) signature is associated with pathologic complete response (pCR) after neoadjuvant chemotherapy in the EORTC cohort. (a) Heat map displaying gene (y-axis) and case clusters (x-axis). The TIL-high and TIL-low groups with respective rates of pCR are indicated. Colors correspond to z-normalized expression levels. (b) Average expression levels ±SEM of each gene in the three primary centroids. (c) Odds ratios (log_e transformed, ±SEM) for probability of pCR derived from Fisher's exact tests for each gene in the TIL signature. The TIL mean represents average expression levels across all eight genes. High and low gene expression categories were assigned using the upper quartile as the cut-point.

Figure 2

Long term outcome of patients according to adjuvant chemotherapy and CD3 status. Representative immunohistochemistry (IHC) staining of CD3 (×40 objective) demonstrating (a) low and (b) dense intraepithelial infiltration of CD3⁺ TIL. (c) Disease-free survival (DFS) of patients treated with anthracycline-based regimens or CMF (cyclophosphamide, methotrexate, fluorouracil) relative to systemically untreated patients in CD3-high (n = 77) and -low (n = 79) subgroups. P_trend, log-rank test for trend.

Figure 3

Additional TIL subsets and their relationship with CD3 status and anthracycline sensitivity. (a) TIL levels in CD3-high and -low cases. Data represent average numbers of TIL expressing CD8, CD4, or CD20 ±SEM. ***P < 0.0001, Mann-Whitney t test. (b) Association of CD8⁺ and (c) CD4⁺ TIL with response to anthracycline therapy. Patients were categorized according to levels of CD8⁺ and CD4⁺ TIL and assessed for disease-free survival (DFS) with respect to anthracycline-based therapy.