The degree of histological differentiation in operable breast carcinomas has long represented one of the best established prognostic factors which have been validated in multiple independent studies [2-4]. The Elston and Ellis modification of the Scarff-Bloom-Richardson grading system separates breast cancer patients into distinct prognosis groups: grade 1, 2 or 3, with a low, intermediate or high risk of recurrence, respectively [5]. Although internationally accepted among pathologists, one of the limitations of the histological grading scheme is that a high percentage (30% to 60%) of breast cancer is still classified as grade 2, a category with ambiguous clinical significance [6].

To be of clinical use, a prognostic factor must show a wide separation in the outcome of the groups identified and select adequate numbers in each group [7]. Notwithstanding many efforts, no single prognostic factor in breast cancer meets these criteria. Histological grading has been combined with tumor size and lymph node stage to form the Nottingham Prognostic Index (NPI), which allows stratification of patients into three different prognostic groups [3] and satisfying these criteria.

Within the last decade, several attempts have been performed to classify grade 2 tumors into two distinct molecular subclasses, improving biological and clinical usefulness of histological grading [8-15]. A multitude of factors, such as HER2, p53, proliferation markers and vascular channel invasion, has been extensively studied and tested in clinical settings, but their importance needs to be validated in statistically robust studies.

Over the last years, laboratory research has proposed novel prognostic markers but not sufficiently investigated to demonstrate their prognostic value. Most of these markers are involved in breast cancer biology and related to essential aspects of cell life, proliferation, transformation and apoptosis [16]. Recently, we have demonstrated that the Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1), an adaptor protein for membrane macromolecular complexes, is a potential candidate of clinical relevance for human breast cancer [17-19]. The strong correlation with poor vascularization and the hypoxia-inducible factor-1α (HIF-1α), a marker of hypoxic tumors, indicates that NHERF1 expression might play an important role in driving metastatic progression by modifying the tumor microenvironment [20]. Among several hypoxia related genes, the vascular endothelial growth factor (VEGF) mediates its effects on proliferation and survival mostly through the VEGF receptor 1 (VEGFR1) and 2 (VEGFR2) within endothelial cells. Nevertheless, it has been demonstrated that VEGFR1 expression in breast cancer cells correlated significantly with high metastasis risk and relapse [21,22]. During malignant cancer progression, TWIST1 plays a role in the development of distant metastasis by inducing an epithelial-to-mesenchymal transition of epithelial breast cancer cells and by prompting them to enter the bloodstream [23,24].

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 [17-19], aggressiveness [21,22], hypoxic response [29,33] and cell invasion/metastasis [24,34], 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 [35,36]. 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 [18,31]. However, in line with several reports describing the NHERF1 gene as transcriptionally regulated by estrogen [18,31,37], 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 [21,22], 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 [1,19]. 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 [44,45]. 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 [17,18] 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.