Molecular alterations in key-regulator genes among patients with T4 breast carcinoma

Since the staging systems of breast cancer were introduced during the course of the last century, the involvement of the skin has always been considered a morphologic characteristic leading to the classification of the tumour into the highest non-metastatic disease stage. In the current edition of the International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) TNM staging system [1], primary breast cancers with extension to the skin are classified as T4. Patients with T4 carcinomas of any type, with or without lymph node involvement, and without distant metastases (T4 N0-2 M0), are classified as disease stage IIIB. According to this system, the breast carcinoma with skin involvement is included in stage III and may be considered as locally-advanced breast cancer (LABC) [1‐3].

In addition to the tumour size and the axillary lymph node involvement, other well-established prognostic factors currently used in breast cancer include histological subtype or grade, estrogen (ER) and progesterone (PR) receptor status, HER2 amplification, and Ki67 proliferation index [4, 5]. Novel tumour markers with potential clinical utility are thus awaited.

The molecular mechanisms underlying locally-advanced breast carcinomas are largely unknown. A distinct gene-expression profile has been described for T3/T4 tumours in comparison to the gene-expression pattern of T1/T2 tumours [6], suggesting that a distinct biological behaviour may characterize initial vs. locally-advanced breast carcinomas. The mitogen activated protein kinase (MAPK) pathway, a major signalling cascade involved in the control of cell growth and proliferation, has been indicated to play a role in the intracellular signalling process of breast carcinomas [7‐9]. The ERK1-2 proteins, which represent the final components of such a signalling kinase cascade, have been found to be activated through phosphorilation (pERK_1-2) in human cancer and implicated in rapid malignant cell growth, mostly as a consequence of mutations in upstream components of the pathway [10, 11]. Presence of pERK_1-2 could be thus considered as a marker for the increased activity of ERK1-2, which may induce cell proliferation, rapid cancer cell growth, and resistance to apoptosis [10]. Moreover, a genomic instability with an increased number of copies of the CyclinD1 gene, which encodes a component of the p16^CDKN2A-RB pathway functionally interacting with the MAPK pathway [12, 13], has been described to promote a deregulation of the cell cycle with subsequent induction of an uncontrolled cell proliferation and tumour growth [14]. Nevertheless, the p53 protein represent the final effector of the p14^CDKN2A-MDM2 pathway; in majority of human cancers, the TP53 gene is functionally inactivated [15]. Lack or reduced expression levels of the p53 protein seems to be associated with a defective apoptotic response to genotoxic damage and, thus, to anticancer agents [16].

Finally, two additional mechanisms seem to play a central role in breast cancer progression and resistance to treatment. The increased expression of survivin, a member of the inhibitor-of-apoptosis (IAP) protein family, has been demonstrated to be associated with resistance to apoptosis [17‐19]. It has been reported that survivin and other IAP proteins cooperate to activate kinase cascades which control cell motility, thus stimulating tumour cell invasion and promoting metastasis [19]. Survivin is indeed overexpressed in most cancer cells and tissues of different histological origin, being correlated to overall survival and acting as a poor prognostic factor in some cancer patients [20‐22]. In breast carcinomas, the up-regulation of survivin has been hypothesized to act as a factor exerting resistance against tamoxifen-induced apoptosis [23, 24]. The second additional mechanism involved in breast cancer pathogenesis includes an increased activity of the human homologue of the Drosophila prune (h-prune), which belongs to a superfamily of phosphoesterases [25]. It has been demonstrated that h-prune is able to promote cell motility through either induction of its phosphodiesterase activity (very recently, a multi-domain adaptor protein, ASAP1, has been reported to stimulate the h-prune phosphodiesterase activity [26]) or interaction with specific protein partners (mainly, nm23-H1) [27‐29]. The h-prune protein has been found expressed at higher levels in breast, colorectal, and gastric carcinomas, participating to the promotion of both tumour invasiveness and metastasis formation [25, 27]. In breast cancer, overexpression of h-prune has been demonstrated to be involved in cancer progression, identifying subsets of patients with higher tumour aggressiveness (although it seems to have no role as independent prognostic factor in clinical outcome of patients with invasive breast carcinoma) [30]. As previously demonstrated [25, 30], gene amplification may play an important role in inducing overexpression of h-prune among breast cancer patients.

In the present study, we examined the expression of survivin, p53, and pERK_1-2 proteins as well as the amplification of CyclinD1 and h-prune genes in a well-characterized cohorts of patients with T4 breast carcinoma and a long follow-up, in order to determine their association with clinical and pathological parameters as well as with patients' outcome.

In this study, we evaluated the impact of some specific molecular alterations (activation of ERK1-2 proteins, amplification of CyclinD1 and h-prune genes, silencing of TP53 gene, overexpression of survivin protein) as predictive and prognostic factors among patients with T4 breast carcinoma. The analyzed molecular alterations have been largely demonstrated to play an important role in: a) deregulating the cell cycle with subsequent induction of abnormal cell proliferation and tumour growth (ERK1-2 phosphorilation and CyclinD1 amplification); b) impairing the apoptotic machinery with subsequent induction of resistance to anticancer agents (p53 downexpression and survivin overexpression); and c) promoting metastasis formation (h-prune amplification). Our findings indicated that subsets of T4 breast cancer patients with pERK_1-2 staining, survivin expression, or h-prune amplification in primary tumour tissues presented a worse overall survival. After multivariate analysis, the pathological response to primary chemotherapy and the survivin overexpression in primary carcinoma represented the main parameters with a role as independent prognostic factors predicting the clinical outcome in such a series of breast cancer patients.

Although an increased expression of survivin in tumour tissues has been already demonstrated to correlate with a poor clinical outcome in a variety of malignancies [20‐22], our results clearly indicated an analogous significant impact on prognosis of such a molecular alteration among T4 breast cancer patients. From the pathogenetic point of view, survivin has been found to provide protection against apoptotic stimuli by inhibiting activation of caspase-9 toward the initiation of the intrinsic mitochondrial pathway of apoptosis [18]. Recently, it has been demonstrated that survivin as well as other members of the IAP protein family are strongly involved in metastasis formation; search for survivin-IAP antagonists may indeed provide new antimetastatic therapies for cancer patients [19]. Nevertheless, survivin seems to be upregulated through the activation of the MAPK-ERK pathway [34]; in other words, the overexpression of survivin may be associated with the increased levels of ERK1-2 phosphorylation (in our series, all 5 cases expressing pERK_1-2 proteins also presented survivin overexpression). Interestingly, our findings indicated that presence of pERK_1-2 expression in primary T4 carcinomas may be indeed correlated with clinical outcome (see Figure 2), suggesting that the cascade of molecular events activating ERK1-2 and upregulating survivin has indeed an important prognostic role in such patients. One could speculate that the lack of a significant association with prognosis for pERK_1-2 staining in multivariate analysis may be due to the fact that we identified only a limited fraction (5/53; 9%) of carriers and, thus, the subgroup analysis relied on a small number of subjects.

The well-established prognostic factors currently used into the management of breast cancer patients include the disease stage as well as the degree of differentiation (tumour grade), the proliferation index, and the hormone receptor status (ER, PR, and, recently, HER2) in primary tumours [35, 36]. In our series of patients with T4 breast carcinoma, no statistically-significant correlation between any of the analyzed molecular alterations and such pathological parameters was inferred. The only exception was represented by the correlation between the pERK_1-2 staining and the Ki67 proliferation index. None of the tumours expressing a high Ki67 proliferation index showed an increased level of pERK_1-2 protein; conversely, all cases with activated ERK_1-2 protein presented a low Ki67 proliferation index. Activation of ERK1-2 proteins has been demonstrated to promote cell cycle progression, participating to induction of cell growth and enhancement of cell survival [10]. Our findings led us to speculate that: a) induction of cell proliferation via pERK_1-2 and Ki67 molecules may represent two unrelated phenomena; and b) among patients with low Ki67 expression levels (who may have an unfavourable prognosis [37], though the role of Ki67 proliferation index as prognostic and predictive marker is yet to be conclusively defined [38]), the presence of pERK_1-2 overexpression seems to identify a subgroup with an even worse prognosis. Taking into consideration the response rates, patients whose tumours had high Ki67 expression levels or HER2 amplification presented the highest rates of response to primary chemotherapy (for Ki67, a significant association was found with both clinical and pathological responses; for HER2, a significant association was surprisingly observed with clinical response only) (see Table 3). These latter findings are consistent with data previously reported [39‐41]. Among the molecular parameters, only pERK_1-2 expression seemed to be significantly correlated with response to primary chemotherapy (significant lower rates were observed for both clinical and pathological responses; see Table 3), reflecting the fact that the activation of ERK1-2 proteins may increase the resistance to apoptosis, reducing the sensitivity to chemotherapy [10].

Several mechanisms have been recently described to participate in progression of breast cancer through activation of the h-prune complex. It is now clear the existence of a network of interacting proteins which indeed regulate the phosphodiesterase activity of h-prune, contributing to promote (ASAP1) or inhibit (nm23-H1) either cancer cell motility and tumour adhesiveness in vitro either tumour invasiveness and metastasis formation in vivo [25‐29]. The increased expression of h-prune protein has been demonstrated to deeply modify this equilibrium of opposite stimuli, playing an important role in promotion of cancer progression [25]. Among others, the main mechanism leading to h-prune overexpression is represented by the amplification of gene copy number [25, 42]. Considering tumours with at least three gene copies, a small fraction (8/53; 15%) of T4 breast carcinomas from our series presented h-prune amplification at chromosome 1q21.3 (see Table 2); such a frequency is quite identical to that described in our previous report (173/1,016; 17%) [30]. All breast cancer patients included into the present study showed axillary nodal involvement; among them, occurrence of h-prune amplification was able to identify a subset with a worse overall survival (see Figure 2). As for pERK_1-2 staining, the low number of events could explain the absence of a significant association of the h-prune amplification with prognosis in the multivariate analysis.

Although our study was retrospective, some important indications about either the prediction of the response to therapy or the role on prognosis in T4 breast cancer patients have been inferred. There is no doubt that the pathological response after primary chemotherapy remains one of the major predictor of survival; however, the molecular marker represented by survivin overexpression may be also considered as a useful prognostic factor in these patients. To validate the incorporation of survivin or the other promising molecular parameters (h-prune and pERK_1-2) as markers for management of T4 patients, further large prospective studies are awaited. Nevertheless, translational studies investigating additional molecular biomarkers should contribute to more accurately identify subsets of patients who would be expected to be more or less likely to respond to specific therapeutic interventions.