Joint Tumor Bud-MMP/TIMP Count at the Invasive Front Improves the Prognostic Evaluation of Invasive Breast Carcinoma

Abstract

Background: Tumor budding is a histological phenomenon consisting of the formation of small clusters of one to five undifferentiated malignant cells detached from the main tumor mass which are observed in the tumor stroma. In the present study, we investigated the prognostic significance of tumor budding in breast cancer and its relationship with the expressions of matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs).

Methods: The number of buds was counted in whole-tissue sections from 153 patients with invasive ductal carcinomas who underwent a long follow-up period. In addition, an immunohistochemical study of MMP-9, -11, and -14 TIMP-1 and -2 expression by cell types at the invasive tumor front was carried out.

Results: There was a wide variability in the number of buds among tumors, ranging from 0 to 28 (median = 5). Tumor budding count ≥ 4 was the optimal cut-off to predict both relapse-free and overall survival. High-grade tumor budding was associated with MMP/TIMP expression by cancer-associated fibroblasts. In addition, we found that the combination of tumor budding grade with MMP/TIMP expression by stromal cells, and especially with MMP-11 expression by mononuclear inflammatory cells, significantly improved the prognostic evaluation.

Conclusion: High-grade tumor budding is associated with a more aggressive tumor phenotype, which, combined with MMP/TIMP expression by stromal cells at the invasive front of the tumor, identifies patients with poor prognosis.

Keywords: MMPs; TIMPs; epithelial-mesenchymal transition; metastasis; tumor invasion.

Figure 1

Breast cancer with low-grade tumor budding (less than 4 tumor buds). (A) Overview (×100) and (B) high-power field (×200). (C) Breast cancer with high-grade tumor budding (more than 4 tumor buds). (D) Overview (×100) and high-power field (×200). Black arrows point to tumor buds.

Figure 2

Distribution of the number of tumor buds in all tumors of 153 patients with breast cancer.

Figure 3

Maximum likelihood determination of the cut-off value of tumor budding count of tumors for predicting relapse-free survival in 153 patients with breast cancer. p-values obtained for each cut-off value are plotted against the value itself. Statistical significance is indicated by the horizontal line at the 0.05 level. (A) Analyses lead to the definition of a count value of four for tumor budding as the optimal cut-off (X² = 19.1; p < 0.0001). (B) Relapse-free survival as a function of the tumor budding count of four as the optimal cut-off for predicting relapse-free survival. Differences in relapse-free survival curves were significant at p < 0.0001. (C) Overall survival as a function of tumor budding count of four. Differences in overall survival curves was significant at p < 0.0001.

Figure 4

Representative examples of the expression of matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs) by stromal cells among buds at the invasive front of breast carcinomas. (A) MMP-9 (×400), (B) MMP-11 (×200), (C) MMP-14 (×400), (D) TIMP-1 (×200), and (E) TIMP-2 (×200). Black arrows point to cancer cells, red arrows point to tumor buds, green arrows to point mononuclear inflammatory cells (MICs), and blue arrows point to cancer-associated fibroblasts (CAFs).

Figure 5

Kaplan–Meier survival curves (relapse-free survival and overall survival) as a function in 153 breast cancer patients, stratified according to tumor budding grade and the expression of MMPs and their inhibitors (TIMPs) by MICs. Tumor budding was dichotomized into low-grade (≤4) or high-grade (>4). Samples on tissue sections were insufficient or lost for analysis in three cases of MMP-9, MMP-11, TIMP-1, and TIMP-2 and in four cases of MMP-14.

Figure 6

Kaplan–Meier survival curves (relapse-free survival and overall survival) as a function in 153 breast cancer patients, stratified according to tumor budding grade and the expression of MMPs and their inhibitors (TIMPs) by CAFs. Tumor budding was dichotomized into low-grade (≤4) or high-grade (>4). Samples on tissue sections were insufficient or lost for analysis in three cases of MMP-9, MMP-11, TIMP-1, and TIMP-2 and in four cases of MMP-14.