Prognostic significance of TRAIL-R3 and CCR-2 expression in tumor epithelial cells of patients with early breast cancer

We conducted a retrospective study of 63 consecutive patients (aged 42–80 years) with a confirmed histological diagnosis of breast cancer who underwent initial surgery at the Hospital Italiano of Buenos Aires, Argentina. Patients were included if they were diagnosed with stage I/II invasive ductal breast cancer according to the International Union Against Cancer TNM classification system [ 16] and ≥10 years after surgery. Exclusion criteria included neoadjuvant therapies, lack of tissue, and another primary tumor. After surgery, all patients were treated with the indicated therapy, depending on their clinical status and the histopathological characteristics of their tumor, which were determined according to the recommendations of the European Society for Medical Oncology [ 17]. The Instituto de Biología y Medicina Experimental and the Hospital Italiano Ethics Committees approved this study, and informed consent was obtained from patients or the relatives of deceased patients, in accordance with the principles of the Helsinki Declaration. Physicians who were unaware of the pathology results acquired clinical information from patients’ medical records, and the anonymity of the data was ensured using a code made available only to the biostatistician.

Breast tissues embedded in paraffin blocks and fixed in 10% neutral-buffered formalin were retrieved from the surgical archives, and 4-μm thick sections were used in the experiments described below.

These tissues were processed and immunohistochemistry was used to determine the levels of ligands and receptors in TEpC and in spindle-shaped stromal cells, not associated with the vasculature, and it was completed as described in a previously work [ 15].

Immunoreactivity was reviewed and scored independently by two pathologists who were blinded to patient outcomes. In uncertain cases, re-evaluation was performed using a double-headed microscope, and staining was evaluated until a consensus was achieved. The agreement in immunohistochemical evaluation between the two observers was 91.77% (Cohen’s kappa coefficient = 0.895). Each sample was assayed in duplicate and was initially examined at 100× magnification followed by observation of five representative fields at 400× magnification along a projected Z-line. Expression levels were evaluated separately for the TEpCs and spindle-shaped stromal cells, not associated to the vasculature, per the percentage of positive cells and staining intensity, which were estimated according to the Allred score [ 15, 16]. The percentages of positive cells were assigned scores as follows: 0 (<10%), 1 (10%–30%), 2 (31%–60%), 3 (61%–90%), and 4 (>90%). Staining intensity was scored as 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong), according to the relative intensity of staining of TEpCs analyzed using the anti-cytokeratin antibody [ 15, 18]. The final staining score was calculated using the sum of the percentage of positive cells and the staining intensity score, which ranged from 0 to 7. Stromal cells included in this study had a spindle shape and were not associated with vasculature. CD34 expression was undetectable in this type of stromal cells as previously reported [ 18].

Classical prognostic markers were categorized according to cut-offs used in the protocols of the Hospital Italiano, [ 17] including: a) age < 50 or ≥50 years; b) tumor size <2 or ≥2 cm; c) histological grade according to the Scarff-Bloom-Richardson grading system [ 19], which is expressed as differentiated (G1), intermediate (G2), and poor (G3); d) expression of estrogen/progesterone receptors and HER2/neu was defined as negative or positive according to Wernicke et al. [ 17]; and e) presence of regional metastatic lymph nodes was recorded as negative (negative nodes in axillary dissection or sentinel lymph node) or positive (including micrometastasis) (Table 1).

To evaluate the statistical significance of the associations between the expression of ligand or receptor and patients’ clinicopathological characteristics, we determined an optimal cut-off value according to a previous study [ 18]. The cut-off value was used to assign protein expression in tumor samples as negative/low or high. To determine the optimal cut-off value, the first quartile (Q1), median, and third quartile (Q3) values were tested individually using univariate analysis and compared with OS. The cut-off value with lowest p value was chosen. The optimal cut-off values for protein expression in TEpCs were as follows: OPG = 6 (Q3), TRAIL =6 (Q3), TRAIL-R1 = 0 (Q1), TRAIL-R2 = 6 (Q3), TRAIL-R3 = 5 (median), TRAIL-R4 = 5 (Q1), RANKL =3 (Q1), RANK =6 (Q1), SDF-1 = 5 (Q1), CXCR-4 = 4 (Q1), IL-6 = 4 (median), IL-6R = 6 (median), CCL-2 = 6 (Q3), and chemokine (C-C motif) receptor 2 (CCR-2) = 6 (Q3). The optimal cut-off values for protein expression in spindle-shaped stromal cells, not associated with vasculature, were as follows: OPG = 2 (median), TRAIL =4 (median), RANKL =2 (Q1), SDF-1 = 2 (Q1), IL-6 = 4 (Q3), and CCL-2 = 3 (Q3). We used Fisher’s exact test to evaluate the association between the expression of these proteins with classical prognostic markers as well as local relapse and metastatic occurrence. Moreover, the association between the ligand and receptor expressions in TEpCs and spindle-shaped stromal cells and metastatic occurrence is displayed as a heat map prepared using Excel.

The expression of TRAIL was significantly associated with lymph node status (Table 2). High TRAIL expression was detected in 10/40 breast cancer patients with negative lymph nodes, and TRAIL expression was undetectable in 0/16 of patients with positive lymph nodes. SDF-1 expression was significantly associated with tumor size and was high in 31/44 patients with tumors <2 cm and in 7/17 patients with tumors ≥2 cm ( p = 0.004, Table 2). High levels of CCL-2 expression were detected in some patients with negative (3/15) or positive (1/42) ER expression (Table 2). The DFS of patients with high CCL-2 expression was 67.7 ± 32.0 months, compared with 123.15 ± 8.28 months for those with low/negative CCL-2 expression ( p = 0.048, Table 3).

IL-6R expression in TEpCs was associated with age (Table 4). Specifically, IL-6R expression was higher in 6/8 patients <50 years of age and in 15/48 patients ≥50 years of age (Table 4). Patients with high expression of TRAIL-R3 and CCR-2 in TEpCs were at significantly higher risk for metastatic tumors than patients with low expression (Table 4). High levels of TRAIL-R3 were expressed in 7/11 breast cancer patients with metastasis and in 12/45 patients with non-metastatic tumors ( p = 0.032, Table 4). Certain patients with metastatic (5/11) or non-metastatic tumors (2/42) expressed high levels of CCR-2 (Fig. 1 and Table 4). There was an association of high TRAIL-R3 expression with shorter DFS, MFS, and OS (Table 3). The values of DFS, MFS and OS of patients with high TRAIL-R3 expression were as follows (months): 90.04 ± 14.64, 97.02 ± 14.08 and 112.75 ± 12.73, respectively; for patients with low/negative expression were 136.22 ± 7.52, 140.22 ± 6.61 and 146.51 ± 5.16, respectively (Fig. 2 and Table 3).

Furthermore, there was an association of high CCR-2 expression with shorter DFS, MFS and OS (Table 3). The values of DFS, MFS, and OS of patients with high CCR-2 expression were as follows (months): 87.57 ± 18.57, 87.71 ± 18.58, and 114.67 ± 15.29, respectively; for patients with low/negative expression were 127.57 ± 8.42, 133.94 ± 7.52, and 140.44 ± 6.41, respectively (Fig. 3 and Table 3).

SDF-1 expression in spindle-shaped stromal cells was associated with histological grades, and high SDF-1 expression was detected in 10/15, 14/21, and 8/24 patients with differentiation grades G1, G2, and G3, respectively (Table 5). In contrast, high expression of OPG and CCL-2 in stromal cells was associated with a higher risk of metastasis (Fig. 1 and Table 5). High expression of OPG was observed in 7/10 patients with metastatic tumors and in 14/43 patients with non-metastatic tumors ( p = 0.038, Fig. 1 and Table 5). In patients with metastatic or non-metastatic tumors, 4/11 and 4/44 expressed high levels of CCL-2, respectively (Fig. 1 and Table 5).

Of clinical variables analyzed, only tumor size was associated with MFS (Table 3). Patients with tumors >2 cm had earlier metastasis compared with those with tumors ≤2 cm as follows (months): 93.00 ± 15.59 vs 139.02 ± 6.47, respectively.

TRAIL-R3 expression in TEpCs was an independent prognostic factor for DFS and OS (Table 6). Moreover, tumor size and CCR-2 expression were independent prognostic factors for MFS (Table 6).