Loss of stromal caveolin-1 expression predicts poor clinical outcome in triple negative and basal-like breast cancers

Abstract

Here, we investigated the possible predictive value of stromal caveolin-1 (Cav-1) as a candidate biomarker for clinical outcome in triple negative (TN) breast cancer patients. A cohort of 85 TN breast cancer patients was available, with the necessary annotation and nearly 12 years of follow-up data. Our primary outcome of interest in this study was overall survival. Interestingly, TN patients with high-levels of stromal Cav-1 had a good clinical outcome, with >50% of the patients remaining alive during the follow-up period. In contrast, the median survival for TN patients with moderate stromal Cav-1 staining was 33.5 months. Similarly, the median survival for TN patients with absent stromal Cav-1 staining was 25.7 months. A comparison of 5-year survival rates yields a similar pattern. TN patients with high stromal Cav-1 had a good 5-year survival rate, with 75.5% of the patients remaining alive. In contrast, TN patients with moderate or absent stromal Cav-1 levels had progressively worse 5-year survival rates, with 40 and 9.4% of the patients remaining alive. In contrast, in a parallel analysis, the levels of tumor epithelial Cav-1 had no prognostic significance. As such, the prognostic value of Cav-1 immunostaining in TN breast cancer patients is compartment-specific, and selective for an absence of Cav-1 staining in the stromal fibroblast compartment. A recursive-partitioning algorithm was used to assess which factors are most predictive of overall survival in TN breast cancer patients. In this analysis, we included tumor size, histologic grade, whether the patient received surgery, radiotherapy or chemotherapy, CK5/6, EGFR, p53 and Ki67 status, as well as the stromal Cav-1 score. This analysis indicated that stromal loss of Cav-1 expression was the most important prognostic factor for overall survival in TN breast cancer. Virtually identical results were obtained with CK5/6 (+) and/or EGFR (+) TN breast cancer cases, demonstrating that a loss of stromal Cav-1 is also a strong prognostic factor for basal-like breast cancers. Our current findings may have important implications for the close monitoring and treatment stratification of TN and basal-like breast cancer patients.

Figure 1

Stromal Cav-1 staining in triple-negative breast cancer patients. Tissue sections cut from formalin-fixed paraffin-embedded triple-negative breast cancer samples were either subjected to H&E staining (a and c) to visualize overall cell morphology, or immunostaining with antibodies directed against Cav-1 (b and d). Note the presence of prominent stromal Cav-1 immunostaining in (b), and the absence of stromal Cav-1 immunostaining in (d). Immunostained sections were counter-stained with hematoxylin. Note that (a and b) are paired images from one representative tumor, while (c and d) are paired images from another representative tumor.

Figure 2

Kaplan-Meier analysis of stromal Cav-1 levels predicts overall survival in triple negative breast cancer Patients. Of the 85 TN breast cancers examined, 83 could be semi-quantitatively scored for stromal Cav-1 levels. Interestingly, 24 patients showed high levels of Cav-1 stromal staining, while 22 showed a lower, intermediate level of staining, and 37 showed an absence of Cav-1 stromal staining. The results of this analysis were highly statistically significant (p = 2.8 × 10⁻⁶). Patients with high-levels of stromal Cav-1 (score = 2), had a good clinical outcome, with >50% of the patients remaining alive during the follow-up period (nearly 12 years). In contrast, the median survival for patients with moderate stromal Cav-1 staining (score = 1) was 33.5 months. Similarly, the median survival for patients with absent stromal Cav-1 staining (score = 0) was 25.7 months (see Table 1).

Figure 3

Kaplan-Meier analysis of epithelial Cav-1 levels does not predict overall survival in triple-negative breast cancer patients. Interestingly, the levels of tumor epithelial Cav-1 had no prognostic significance (p = 1.9 × 10⁻¹). In this cohort, 85 patients were scored, with 50 patients showing an absence of epithelial Cav-1 staining, and 33 patients showing moderate-to-strong epithelial Cav-1 staining. Median survival rates were 35.4 months [epithelial Cav-1 (−)] versus 46.5 months [epithelial Cav-1 (+)]; 5-year survival rates were 30.95% [epithelial Cav-1 (−)] versus 41.45% [epithelial Cav-1 (+)] (see Table 4).

Figure 4

A comparison of stromal Cav-1 staining with other risk factors in triple negative breast cancers. A recursive-partitioning algorithm was used to assess which factors are most predictive of overall survival in TN breast cancer patients. In this analysis, we included tumor size, histologic grade, whether the patient received surgery, radiotherapy or chemotherapy, CK5/6, EGFR, P53 and Ki67 status, as well as the stromal Cav-1 score in this predictive algorithm. There are essentially three risk groups identified in this model: (1) Low-risk, high stromal Cav-1 staining (score = 2); (2) Medium-risk, low stromal Cav-1 staining (score = 0 or 1), with low-histologic grade (I or II); and (3) High-risk, low stromal Cav-1 staining (score = 0 or 1), with high-histologic grade (III or IV). For the low-risk group, >50% of the patients remained alive during the follow-up period (nearly 12 years). For the medium-risk, and low-risk groups, the median survival was 69.8 months, and 25.6 months, respectively (see Table 5). This analysis indicates that stromal Cav-1 expression is the most important prognostic factor for overall survival in TN breast cancer (p < 0.001).

Figure 5

Kaplan-Meier analysis of stromal Cav-1 levels predicts overall survival in basal-like triple-negative breast cancer patients. We also determined the prognostic value of stromal Cav-1 in basal breast cancer patients. For this purpose, we selected the TN patients who stained positively for either CK5/6 or EGFR, for inclusion as basal-like breast cancer patients in this analysis. Using this approach, 57 of the TN breast cancer cases were re-classified as basal-like breast cancers. Note that Kaplan-Meier analysis of stromal Cav-1 status in basal-like breast cancer patients was highly statistically significant (p = 2.2 × 10⁻⁶). As such, stromal Cav-1 status also has strong prognostic significance in TN patients with the basal-like breast cancer phenotype.

Figure 6

Kaplan-Meier analysis of stromal Cav-1 levels predicts overall survival in basal-like triple negative breast cancer patients. As in Figure 5, except that patients that recieved a stromal Cav-1 score of 1 or 2 were considered as a single group (See 1 + 2), and then compared with patients that received a stromal Cav-1 score of 0. Note that Kaplan-Meier analysis of stromal Cav-1 status in basal-like breast cancer patients was highly statistically significant (p = 1 × 10⁻⁶).