Clinical and pathological factors, such as nodal status, tumor grade, proliferative activity, receptor status and HER2 overexpression, are currently used for determining the risk of relapse of breast cancer patients. Traditional prognostic factors have shown limited ability to predict distinct patient outcomes and individuals with the same clinical assessment can have markedly different courses [32
]. Prognostic heterogeneity is complicated by a myriad of liable alterations within multiple biological pathways, stressing the need for further studies on molecular events involved in cancerogenesis, tumor progression and metastasis.
In this study, we explored traditional prognostic factors and a panel of tumor markers not used in routine diagnosis, such as NHERF1, VEGFR1, HIF-1α and TWIST1, that have been respectively related to breast cancer progression [17
], aggressiveness [21
], hypoxic response [29
] and cell invasion/metastasis [24
], assessing if they are differentially expressed in tumors scored as grade 2, in order to best characterize them. We showed that several parameters discriminated poor versus good clinical outcome on 187 patients, but the PVI together with MIB1 were the two variables that remained strongly associated with worse prognosis in multivariate analysis. Our results are consistent with numerous findings, which highlight proliferation-related genes as the main and common denominator for predicting clinical outcome [35
]. However, it is not surprising that these genes are involved in breast cancer prognosis, considering that the increased rate of proliferation and defects in cell-cycle regulators are two of the features common to breast carcinomas.
In the present study, overexpression of cytoplasmic NHERF1 resulted associated with unfavorable prognosis and aggressive clinical parameters, as previously demonstrated [18
]. However, in line with several reports describing the NHERF1 gene as transcriptionally regulated by estrogen [18
], here we demonstrated that cytoplasmic NHERF1 expression was significantly correlated with increasing ER expression. Interestingly, we also observed a significant direct correlation between increased levels of cytoplasmic NHERF1 and VEGFR1. Tumors overexpressing NHERF1 and VEGFR1 revealed an association with poor outcome, being characterized by an increasing tumor grade, and negative status of steroid hormone receptors. It is now clear that NHERF1 promotes dimerization and activation of many tyrosine kinase receptors, such as the platelet derived growth factor receptor [38
], the epidermal growth factor receptor [39
] and the HER2 [17
], known to directly regulate cell pathways related to cancer progression. Previously, we observed that VEGFR1 was expressed in the cytoplasm of breast cancer cells, where also NHERF1 expression was predominant [19
]. In this context, NHERF1 acts as a potential interacting partner of VEGFR1, a marker correlated with high metastasis risk and relapse [21
], probably promoting invasion of tumor cells through autocrine and paracrine mechanisms [40
In our series, tumors of grade 2 represent 46% of the whole cohort, confirming previous observations that this constitutes a substantial proportion of cases in current routine breast cancer diagnostics [41
]. The Elston and Ellis modified grading system provides a simple, inexpensive and routinely applicable overview of the intrinsic biological characteristics and clinical behavior of tumors [4
]. Grade 2 tumors usually show an intermediate outcome during the early years of follow-up, leading to speculate the uncertain role of histological grade in therapeutic planning due to the not informative results for clinical decision making [6
]. Further sub-classification of grade 2 tumors, possibly into low and high risk categories, would be beneficial to improve prognostic stratification of these patients. Notably, we found that grade 2 tumors exhibited significant correlation with parameters of low-risk significance, such as small tumor size, lower nodal stage and proliferative activity and positive steroid hormone receptor status. Afterwards, we investigated what traditional clinicopathological parameters and new potential markers, such as NHERF1, VEGFR1, HIF-1α and TWIST1, could best characterize grade 2 tumors with poor prognosis. Our results demonstrated that a distinct immunophenotype, PVI/membranous NHERF1, is able to categorize grade 2 tumors into two defined subgroups, which exhibited significantly different prognosis. Interestingly, 72% of grade 2 tumors with the PVI+/membranous NHERF1- expression phenotype were associated to an adverse prognosis. However, a significant high proportion of grade 3 tumors showed the same PVI+/membranous NHERF1- expression phenotype, highlighting that grade 2 tumor subgroup with poor prognosis is regarded as being similar to grade 3 cancers. Relevantly, also in the whole cohort the PVI+/membranous NHERF1- expression phenotype displayed a significant correlation with poor prognosis tumors. Since, according to these results, the PVI+/membranous NHERF1- expression phenotype in grade 2 tumors is a poor prognosis factor, we have analyzed PVI/membranous NHERF1 immunophenotypes in a subgroup of patients stratified as having a family history of breast cancer, a category with high biological malignancy, as previously notified [1
]. Intriguingly, it was emerged that familial tumors with grade 2 were prevalently associated to the PVI+/membranous NHERF1-expression subset, confirming one more time the prognostic relevance of this tumor immunophenotype.
The pathological significance of the PVI, a marker of tumor with metastatic potential and a predictor of breast cancer outcome, has long been appreciated. In the 9th St Gallen meeting, the presence of vascular invasion was included in the category of relevant prognostic factors in lymph node-negative patients [42
]. Efforts to detect early metastatic activity, such as diligent pathological examination of sentinel lymph node biopsies would be complimented by the objective evaluation of vascular invasion status of the primary tumor [43
]. Vascular invasion has been recently reported as a histoclinical parameter independently associated with poorer survival inside both basal and triple-negative breast cancer phenotypes [44
]. However, in a our previous work, it has been demonstrated the predictive significance of the PVI for familial patients with BRCA gene mutation risk [27
]. Thus, on the basis of our results, the PVI assessment might provide additional information on disease evolution of grade 2 tumors.
The adaptor protein NHERF1 shows a physiological localization at the plasma membrane, but during breast cancerogenesis progressively loses its apical localization becoming mostly cytoplasmic in no longer polarized tumor cells [17
]. In our series, 13% of tumors showed NHERF1 still localized in the plasma membrane, and were positively associated with favorable prognosis parameters, such as low tumor grade, positive PR status, and low proliferative activity. The positive prognostic impact of membranous NHERF1 is in agreement with results obtained from our [17
] and other laboratories [46
], suggesting that NHERF1 might behave either as a tumor suppressor, when it is localized at the plasma membrane, or as an oncogenic protein, when it is shifted to the cytoplasm, depending on its subcellular distribution.
Moreover, in the present study the good prognostic relevance of membranous NHERF1 has been demonstrated both in the whole cohort and in subgroup of grade 2 tumors. From a clinical perspective, the PVI+/membranous NHERF1- expression phenotype could improve the accuracy of predicting clinical outcome for a subgroup of patients. Therefore, the current results indicate that the combination of those two markers may be applicable as predictive markers to select patients for more aggressive treatment and follow-up.