DISCUSSION

Several biomarkers assessable by IHC techniques have been studied as prognostic factors in non-metastatic TNBC, as a single factor, in co-expression, or in a panel of multiple biomarkers.32,33 Well-known biomarkers, including cytokeratins (CK5/6, CK14, and CK17), EGFR, and the c-Kit receptor, are surrogates for the poor-prognostic basal-like subtypes, which represent 50%–80% of TNBC cases.34 Of recent interest are tumor infiltrating lymphocytes and androgen receptors (AR).35,36 Taken one by one or in association, either for the consecrated biomarkers or for the less well-known ones, the prognostic role is controversial.

Thus, Ki67, a non-histone nuclear protein measure of proliferation, is associated with a high probability of relapse and poor survival in early-stage breast cancer patients, regardless of pN status or adjuvant treatment.37,38 High levels of Ki67 were found to be directly related to TNBC tumor grade and size, and correlated with a higher rate of death.39 In our study, Ki67 was significantly correlated with the TNBC phenotype, the mean value being 44.7% vs 22.2% in non-TNBC patients. Ki67 was also correlated with advanced histopathological stage (P=0.004) and nodal involvement (P=0.033), with no difference in survival between patients with a score ≤20% in comparison with those >20%.

EGFR overexpression, one of the four members of the ErbB tyrosine kinase membrane receptor family that, through the Ras-Raf-MEK-ERK pathway regulates tumor cell growth, motility, and apoptosis,40reaches 50% in TNBC (higher than in other breast cancer types) and approaches 90% in the basal-like subtype.34 Previous studies have found correlations between EGFR and poor prognosis features, such as large tumor size and high histopathological grade.41 In a retrospective study of 284 early-stage TNBC patients, EGFR positivity significantly correlated with poorer disease-free (DFS) and overall survival (OS) (HR =2.22; P=0.01 and HR =2.19; P=0.03, respectively).42 In our analysis, a borderline difference existed between EGFR expression in the TNBC and non-TNBC populations, with a paradoxical higher percentage of positivity in the latter subgroup. In TNBC patients, EGFR was more expressed in ductal carcinoma and was associated with shorter EFS compared with EGFR-negative patients (80 vs 92 months; P=0.792).

The transmembrane glycoprotein E-cad, encoded by the CDH1 gene, mediates epithelial cell-to-cell adhesion and plays an important role in cell proliferation and metastasis suppression.43,44 E-cad negativity represents a surrogate marker for a lobular subtype of invasive breast carcinoma, but the absence of expression in non-lobular histology was found to be associated with large tumor size, metastatic lymph nodes, poor tumor differentiation, and the triple-negative phenotype.45,46 E-cad downregulation is a part of the epithelial-to-mesenchymal transition mechanism associated with cancer invasiveness and metastasis reported in the claudin-low TNBC subtype, which has a high relapse rate.47,48 Thus, in 123 TNBC patients primarily treated by surgery, E-cad-negative cases displayed poorer outcomes regarding OS (P=0.0265) and DFS (P=0.0125).31 In another analysis (N=182), E-cad was preferentially negative in the basal-like subtype.49

In our study, negative E-cad staining was recorded in 38.6% (17 patients) of the TNBC population compared with 20.9% (27 patients) in the non-TNBC patient group (P=0.020). E-cad positivity was inversely correlated with tumor grade (P=0.016) and shows a tendency toward a longer mean EFS (85.74 vs 79.10 months in negative staining cases; P=0.797).

Another biomarker apparently more specific than EGFR used to define the basal-like subtype is the high molecular weight basal CK5/6, whose detection by IHC is challenging because of focal and weak reactivity and no precise cutoff, with the percentages of positivity in TNBC varying in the literature from 24% to 72%.50,51 CK5/6 correlates with necrosis and lymph node involvement.52,53 The overexpression of CK5/6 seems to correlate with HER2, Ki67, and p53 expressions.54 In a retrospective study of a non-metastatic population (N=97), the recurrence risk and mortality rate were 2.41-fold (P=0.027), but 3.74-fold higher (P=0.01) in CK5/6-positive patients.55 Conversely, in a small sample (N=53) of TNBC patients, CK5/6 expression was associated with more favorable outcomes than CK5/6 negativity (HR for RFS =0.23, P<0.01; HR for OS =0.39, P=0.02).56

In our study of TNBC patients, CK5/6 expression was correlated with the clinical stage (P=0.010), and there was no correlation with lymph node involvement. CK5/6 was the only biomarker associated with relapse (P=0.006), and the estimated mean EFS in CK5/6-negative patients (68.84 months) was inferior to that of CK5/6-positive patients (98.84 months) (P=0.038).

TP53 is a tumor suppressor gene that encodes the p53 protein and participates in cell cycle progression, apoptosis regulation, and DNA repair.57 Missense mutations can lead to stabilization of the p53 protein that is oncogenic and accumulates in the cytoplasm, thus becoming detectable by IHC techniques.58,59 In breast cancer, the frequency of mutation has been reported to be as high as 30%, whereas in the triple-negative phenotype, it reaches 70% and about 95% in the basal-like subtype.60–62 p53 positivity is directly correlated with lymph node involvement, tumor size and grade, lymphatic invasion, and EGFR overexpression.62,63 High p53 levels are also associated with a high Ki67 score.64

p53 overexpression was found in most analyses to be associated with poor outcomes in TNBC patients.65 However, in a small sample of early-stage TNBC patients (N=32), p53-positive status was an independent predictive factor for a shorter survival span (5.4 relative risk for RFS; P=0.013).66 In other ways, p53 microarray positivity was associated with a better survival rate (142 node-negative TNBC patients), the HR for DFS being 0.52 (95% CI: 0.27–0.97; P=0.004), with a 20.9% relapse rate vs 37.5% in p53-negative patients.67

In our analysis, p53 was one of the few biomarkers with a significantly different expression (P<0.0001) in TNBC patients compared with non-TNBC patients (66.2%, 43 patients vs 32.5%, 113 patients). The risk of relapse was higher, albeit not significantly, in p53-negative patients (HR =1.99; 95% CI: 0.699–5.690; P=0.197). As it is unclear whether p53 expression reflects the wild-type or mutant TP53, our results confirm those of others.

Among the less assessed biomarkers in breast cancer is the Bcl2 protein, an antiapoptotic protein, by participates to the G0 cell cycle prolongation.68 Bcl2’s tumorigenic potential of inappropriate expression is in contrast to human follicular B cell non-Hodgkin’s lymphoma and is associated with improved outcomes in hormone receptor-positive node-negative breast cancer, as it is one of 21 genes validated in the prognostic signature, Oncotype DX (Genomic Health Inc, Redwood City, CA, USA).69,70 In a large cohort analysis of 11,212 women with early-stage breast cancer, Bcl2 expression was found to be a powerful positive prognostic marker in the ER-negative subgroup, independent of adjuvant therapy (HR =0.63; P=0.001).71 In 635 early-stage TNBC patients, Bcl2-negative status was associated with a double risk of recurrence (HR =2.45; P=0.00002) and significantly correlated with high tumor grade and high levels of HER3 and E-cad expression (P<0.001).72 Conversely, in a small cohort of triple-negative (N=124) vs non-triple-negative (N=458) patients, Bcl2 expression was a predictor for poor survival (P=0.028).73 Our Bcl2-negative patients in the TNBC group had a poorer outcome (78.79 months; 95% CI: 63.64–93.95) than those in the Bcl2-positive group (86.43 months; 95% CI: 72.91–99.95), although the difference did not reach statistical significance (HR =1.19; 95% CI: 0.42–3.39; P=0.746).

TOP2A protein catalyzes the breakage and reunion of double-stranded DNA, and its tumor cell staining is correlated with TOP2A gene aberrations, situated on chromosome 17q12-q21.74 Few studies address the question of its prognostic significance in TNBC and the results are inconclusive.75 TOP2A expression was found to be twice as high in breast tumors expressing basal cytokeratins and correlated with a high tumor grade and high Ki67 score.76 Along these lines, in one retrospective study of 145 early-stage TNBC patients, TOP2A positivity was marginally associated with a shorter time to progression (HR =2.01; P=0.059).77

In our 62 TNBC tumor specimens assessed for TOP2A expression, a direct correlation was found with the in situ pattern (P=0.003). Survival without relapse seemed shorter in TOP2A-positive than in TOP2A-negative patients (mean EFS, 74.86 vs 93.21 months; P=0.151).

The Cox proteins (prostaglandin H2 syntheses) are membrane-bound enzymes and members of the mammalian heme-dependent peroxidase family that catalyze the conversion of arachidonic acid released from membrane phospholipids to prostaglandin H2 and reactive oxygen species.78 Cox-2 is normally found at a very low level in most tissues and is highly inducible by tumor promoters. It is amplified in breast carcinoma cells in about 30% of cases, appearing to be correlated with ER expression, lymph node involvement, high histological grade, and, in some studies, HER2 overexpression and high Ki67 or p53 scores.79–81 In ER-negative breast tumors, Cox-2 appears to play a role in neo-angiogenesis, and thus its expression is considered to be a negative prognostic factor.82In our TNBC patients, Cox-2 expression was inversely correlated with capsular effraction (P=0.046), thus appearing as a favorable prognostic factor. Hence, the mean estimated EFS was lower in Cox-2-negative patients, that is 79.35 months compared with 90.41 months in cases expressing the biomarker (P=0.547).