Advances in Molecular and Immunohistochemical Detection of Prognostic and Therapeutic Markers in Breast Cancer

In the last two decades, new discoveries concerning on breast cancer have contributed to important changes on its classification, from purely morphologic to molecular embased, to establish better correlation with clinicopathologic features. The classification in molecular subtypes, based on hormonal receptor and HER-2 status, have been remarkable not only for its more accurated clinical correlations, but also for its easy applicability in diagnostic routine, better replication of tumor microenvironment through the selection of paraffinized tumor amounts and cost-effectiveness of the detection method, the immunohistochemistry. Hence, this classification may predict the breast cancer prognosis and became an important target for therapy with hormonal and HER-2 antagonist drugs. Other study models, like cancer-stem cell hypothesis and immunological aspects of human cancer, have brought new emerging ideas regarding on molecular pathways and accurated prognostic preditions. Putative stem-cell markers and PD-1/PDL-1 , have highlighted among several emerging molecular markers because of the bad cancer prognosis determinated by stem-cell markers expression and for emerging new drugs with selective action to PD-1/PDL-1, with promising results. The therapy of breast cancer have became diverse, target directed and personalized, in order to take in consideration the clinicopathologic cancer aspects, molecular tumor profile and clinical status of the patient.


Introduction
Breast cancer is the one of the three most frequent human neoplastic disease worldwide and is the most common female cancer, remaining with considerable impact on general mortality. Worldwide, in the last 10 years, the incidence is growing up, with approximately 2.1 million of new cases per year and estimated mortality of 15%, at about 300.000 per year [1,2].
Breast cancer remains as an heterogeneous group of disease from the point of view of biological behavior, therapeutic issues and prognostic features, determining different tracks of overall and free of disease survivals [3,4]. Thus, the clinicopathologic classification of breast cancer has been challenging over the last years, since the isolated simple morphologic classification of the tumor on histology examination is not necessarily related to the precise biologic behavior of the disease [5,6].
In this way, especially over the last two decades, important researches revealing novel molecular markers expressed by cancer cells has been published in the literature. The new discoveries have improved the breast cancer classification, which has been progressed from a purely morphologic classification, based on histologic patterns, to a molecular classification, based on expression of oncoproteins and hormonal receptors, detected mainly by immunohistochemical techniques, in paraffin-embedded tumoral specimens [6,7].
The novel molecular classification of breast cancer seems to exhibit more accurated correlation to the clinicopathological aspects of the tumor, as proliferative index, invasiveness and potential to metastatic spread. Furthermore, some of these molecular markers allowed the development of new drugs with specific actions on populations of cancer cells with specific genes alterations, improving considerably the therapeutic, prognostic and survival issues [7].
Instead of the recent advances on new therapeutic protocols under a new molecular perspective, early breast cancer on clinicopathological classification still remains the single one potencially curative [8]. The management of advanced clinicopathologic stage tumors and some established molecular groups of cancer, especially the 'triple negative' disease, remains with lacks of consensus. Anyway, the molecular markers have just improved the pathophysiology pathways knowledge, with potential future development of promising new drugs for target therapy of breast cancer [8][9][10] .

The molecular subtypes of breast cancer of clinicopathologic importance
In the beginning of the 21st century, breast cancer was classified mainly on histologic basis. The WHO current histologic classification of breast cancer is demonstrated in Table 1. Photomicrographies of the most frequent histologic subtypes of invasive breast cancer are represented on Figure 1. The hormonal status receptors (estrogen and progesterone) expression by the neoplastic cells was just evaluated by immunohistochemistry on paraffin-embedded specimens of tumor (core needle biopsy or the surgical excision specimen) [6,7] .
Breast cancer is known for its heterogeneous behavior [3,4]. The histologic classification has been satisfactory for malignancy determination [6]. Though, the clinical division based on hormonal status was not enough for accurate prediction of the prognosis and of clinical response to the therapy [5]. Thus, until the last decade of 20 th century, the clinical treatment of breast cancer was based on unespecific chemotherapy and hormonal therapy with drugs like tamoxifen, a known hormonal receptor antagonist [12].
The hormone positive breast cancer is more "differentiated" than the negative one, as the cancer cells maintain the epithelial original cell feature of hormonal receptor expression and, therefore, the hormonal antagonist drugs are effective against these tumors [8]. On the other hand, the approaching of hormonal negative cancers were variable, since it was forming a kindly heterogeneous group, with different aggressiveness potentials, imprecise therapeutic response and doubtful prognosis [6,8,10].
In the first decade of the current century, it was emerged a promising classification of breast cancer, proposing a division of the disease in 3 molecular subtypes: luminal, HER-2 overexpressed and "triple negative" ( Table 2). This new classification has demonstrated better correlation with the breast cancer behavior. Thus, it was adopted on diagnostic routine of breast cancer. Since this study was published, besides of evaluate the histologic patterns and report the pathologic tumor stage, the pathologist has been required to determine the molecular cancer profile, which has became indispensable to therapy planning [12,13]. The luminal subtype cancer is the hormonal positive tumors. This kind of cancer is frequently well or moderately differentiated on histology, formed by lower grades of cells, with lower proliferative index, which is evaluated by antibody Ki-67/MIB-1 on immunohistochemistry. The majority of breast cancers are classified as this subtype (Figure 2). Eventually, luminal cancer can overexpress or amplify at the same time the protein called human epithelial growth factor receptor 2 (HER-2), codified by the oncogen ERBB2 [14,15].
ERBB2 is a oncogen localized in chromosome 17, which codifies the HER-2 protein, a type I transmembrane protein with an extracellular and an intracellular domains, activating signaling pathways from extracellular signals. In last instance, the overexpression/amplification of HER-2 overactivates the intracellular protein kinases, dysregulating the cell cycle, disrupting the cell adhesion and cell polarity and promoting the invasive phenotype [16].  The breast cancer classified as HER-2 subtype is necessarily negative for hormonal receptors and is featured by overexpression or amplification of HER-2. This subtype is frequently less differentiated than the luminal ones on histology, constituted by high grades of cancer cells, with high proliferative index. The presence of elevated concentration of intratumoral lymphocytes (TIL) is not an uncommon finding in these tumors [17].     This new receptor was one of the pioneers for target therapy in molecular era of breast cancer approaching, as it was developed a new class of drug, called trastuzumab, with selective action against the cancer cells overexpressing/amplifying HER-2. Besides the HER-2 subtype tumors, this drug is also recommended for the luminal ones with positive status for HER-2 [18,19].

Photomicrographies of hematoxylin & eosin (H&E) slides illustrating the most frequent histologic subtypes of infiltrating (invasive) breast cancer. (A) Infiltrating duct carcinoma (Invasive ductal carcinoma NOS) is the most frequent histologic subtype of breast cancer (nearly 75-80% of all invasive breast cancer), constituted of cohesive cancer cells forming infiltrative ductal and ribbons structures (4×); (B) Lobular invasive carcinoma is the second most frequent invasive breast cancer (5-15% of all invasive breast cancer), composed of infiltrating cancer cells with diffuse single-file pattern (10×). In this subtype, the cancer cells lose the cohesion (e-cadherin, an immunomarker important for cell adhesion evaluation, is negative on immunohistochemistry); (C) Mucinous carcinoma represents approximately 2% of breast invasive cancer, composed of groups of cancer cells outling ductal structures, immersed in mucin pools
The status of HER-2 expression is analyzed through immunohistochemistry of paraffin-embedded specimens of the breast cancer (Figure 3a). The tumor is considered negative for HER-2 if it is not labeled (score zero) or the cell membrane is partially labelled for the HER-2 antibody (score 1+). The tumor is positive for HER-2 if all the cell membranes outlines are strongly labeled for this antibody (score 3+). Finally, in part of the cases, the HER-2 antibody can label totally the cancer cell membrane, but with low intensity or can label partially the cell membrane with high intensity. In these situations, the HER-2 status is considered equivocal (score 2+). The confirmation of overexpression/amplification must be evaluated through fluorescence "in situ" hybridization (FISH) (Figure 3b) [21,22].
The "triple negative" breast cancer is negative for hormonal receptors and HER-2. It is the less differentiated tumor subtype on histology, formed by highest grades cancer cells, with highest proliferative index, presenting the worst prognosis among the 3 molecular subtypes. This tumor still does not present an specific therapy, which is chosen depending on the clinicopathological stage. In metastatic disease, the treatment focuses on quality of life and palliation. In "triple negative" tumors, the evaluation of BRCA status is mandatory [8,21].

Germline mutations of BRCA-1/BRCA-2 genes: increased risk of breast cancer development during the life
Identified in 1994, BRCA-1/BRCA-2 are tumoral suppressor genes, respectively located in chromosome 17 and 13. Mutations of these genes are related to hereditary breast cancer, estimated in 5-10% of all breast malignancies. BRCA-1/BRCA-2 play a central role in DNA repair [23,24]. Mutations of these genes increase the susceptibility for DNA damages. "Triple negative" subtypes carry more frequently mutations of BRCA-1 and mutations of BRCA-2 increase the risk for luminal subtypes of breast cancer. HER-2 overexpression is inversely correlated to BRCA mutations [24,25].
It was observed in some studies that "triple negative" breast cancers with BRCA mutations present more chemosensitivity than the ones without BRCA mutations. Chemotherapy with DNA-damaging drugs, like the alkylating agents and anthracycline, can prolong the free of disease survival for tumors of triple negative phenotypes. This found is expected, since BRCA mutation prejudices the DNA repair and, consequently, increase the sensibility to DNA damages of cancer cells by these drugs. Neither therapeutic response nor free of disease survival of luminal subtypes of breast cancer seems to be influenced by BRCA mutations [8,24,26].
Regarding on prognosis, multiples studies present conflicting results. The prognosis depends on tumor features, especially the molecular subtypes and the clinicopathologic stage. The predictive value depends on the administrated therapy. Thus, BRCA-1 mutated breast cancer probably present worse prognosis than the BRCA-2 mutated ones, since BRCA-1 mutated tumors are mainly of "triple negative" phenotype, therefore intrinsically more aggressive than the luminal subtypes harboring BRCA-2 mutations [24,27].
The tumoral suppressor proteins codified by BRCA-1/BRCA-2 act on homologous recombination repair of double stranded DNA breaks. Homologous recombination mechanism protect the integrity of genome in proliferating cells. BRCA-1 recognize DNA damage and recruit DNA repair proteins. BRCA-2 mediates the recruitment of another protein, called RAD51, to double stranded DNA breaks, allowing for homologous recombination repair [24,28].
In BRCA-mutant breast tumors, the base excision repair pathway is important for cancer cell survival, in response to single stranded DNA breaks. Polyadenosine diphosphate-ribose (PARP) is a family of DNA repair enzymes, playing a key role in base excision repair mechanism. These enzymes are recruited to the site of DNA damage and add ADP-ribose to target nuclear proteins, causing post-translational modifications and restarting stalled DNA replication. BRCA-mutant breast cancer presents deficiency of homologous recombination repair, with overactivated PARP, leading the cancer cell to avoid apoptosis [24,26,28].
The inhibition of PARP cause persistance of single stranded break, resulting in stalled replication and double strand breaks. This mechanism leads to accumulation of DNA damage, causing cell cycle arrest and apoptosis. The PARP inhibitors form an emerging class of drugs, which have been recommended to chemotherapy for BRCA-mutant breast cancer and empirically for metastatic breast cancer, with promising results [24,25,28].

Cancer stem-cell hypothesis: impact in breast cancer prognosis
In the last two decades, experimental evidences in several studies of neoplastic tissues have revealed a population of cancer cell with properties of self-renewal, differentiation to multiple lineages ability and low proliferative index. These properties have been considered cancer stem-cell like features and attributed to a possible cancer stem-cell lineage present in the tumor bulk [29,30].
Cancer stem-cell has awaked interest in the context of breast cancer because of its characteristic heterogeneity of biological behavior and therapeutic response. It has been hypothesized that cancer stem-cell might be one of the causes of the high variability of biological and prognostic spectrum of breast cancer. Cancer-stem cells might play an important role on therapeutic resistance and progression of disease, affecting the overall and free of disease survival [31,32].
Thus, an important feature which allows possible cancer stem-cell resistance to chemotherapy is its low expression of surface proteins. Because of its self-renewal properties, cancer stem-cell does not depends on signaling from other cells to proceed its functions in tumoral tissues. Furthermore, for its low antigenicity and low proliferation index, there are few alternatives for drug interactions. DNA damage agents are poor effective against these cells possibly for a lack of proliferation, as well new classes of drugs, like PARP inhibitors, which better act on cells in proliferative phase [31,33].
One of possible pathways for breast cancer therapeutic resistance acquired along the time might be explained by populations of cancer stem-cells not eliminated, selected by multiple chemotherapy cycles. Tumoral cells in active proliferation phase are more hitten, increasing the proportion of indolent cells with stem-like features in cancer cell population. Through the capacity of multilineage differentiation, cancer stem cells might generate new daughter cells with more aggressiveness and chemoresistance [32,34].
The identification of cancer stem-cells is challenging. First, because of its irregular distribution in selected tumor amounts. Second, for definition, these cells are frequently scarces in tumor bulk. In this way, these cells are better identified through "in vitro" methods, like cellular cultures. However, the mainly disadvantage of this technique is the fact of stem cells behave in a different fashion in artificial environment, since the cell phenotype expression depends on their interactions [32,35].
Advances in Molecular and Immunohistochemical Detection of Prognostic and Therapeutic… DOI: http://dx.doi.org /10.5772/intechopen.94462 Thus, several studies with cancer stem-cells in different neoplastic tissues have been accomplished with conflicting results. An interesting method to identify these cells in their original environment is the immunohistochemistry performed on amounts of paraffin-embedded neoplastic tissues, with the advantages to allow the evaluation of phenotype expression next to the reality and to be easily performed and cost-effectiveness in diagnostic routine [35].
In the last years, some putative stem-cell markers detected by immunohistochemistry have been tested in paraffinized tissues of breast cancer. Multiple studies have demonstrated that expression of putative stem-cell markers by tumoral cells seems to worse the prognosis and survival in breast cancer. The most frequent studied stem-cell markers are CD24, CD44, CD133 and EPCAM, with two identified putative stem-cell phenotypes: CD24 low/CD44 enriched and co-expression of CD133 and EPCAM (Figure 4). Besides of the scarcity of stem-cells in neoplastic tissues, the conflictous results of these studies might be explained by a necessity to qualitative analysis of these markers expression, exactly for the rarity of stem-cells [32,36].
In some studies, identification of a stem-cell like phenotype CD24 low/CD44 enriched have prejudiced the free of disease survival, especially in cases of early stages of breast cancer, with more occurrence of distant metastasis and cancer recurrence after surgical and adjuvant treatments. The presence of cancer cells with positivity for cancer stem-cell phenotype CD133/EPCAM is has been related to poor overall survival in breast cancer, with more adjuvant therapeutic fail [32].
For the moment, these putative stem-cell phenotypes seems to be independent prognostic factors in breast cancer. "Triple negative" breast cancer and BRCA-1 mutant breast cancer have been associated to stem-cell like phenotype CD24 low/CD44 enriched. These putative stem-cell markers may become possible future targets for new drugs in the future [30,32].

Immunologic aspects related to breast cancer
In the context of cancer, the immune system can suppress the tumor growth by the destruction of cancer cells or inhibition of their outgrowth. On the other hand, immune system can play a role on tumor progression by the selection of tumor cells which are adapted to survive in an immunocompetent host or modifying the tumor environment to facilite the tumor outgrowth [37] .
Elevated levels of CD4+ regulatory T lymphocytes (Tregs) found in many cancers are associated to poor prognosis. Tregs create a favorable immunosuppressive microenvironment to the outgrowth and progression of the tumor. On this way, FOXP3 is expressed by the Tregs and can be detected by immunohistochemistry. FOXP3 is responsible for induction and maintenance of tolerance to self antigens in normal cells, as well this immunotolerance can be performed by the Tregs with cancer cell antigens [37,38].
Another example of cancer cell escape mechanism from the immune system is caspase-8 mutations present in "triple negative" breast cancers and other solid malignant tumors. These mutations abolish the death induced by cytotoxic lymphocytes CD8+ in tumoral cells [37,39].
The activation of T lymphocytes by foreign antigens occurs by concomitant major histocompatibility complex (MHC) antigen presentation and co-expression of T-cell receptor (TCR). At the same time, a family of T-cell transmembrane proteins CD28/B7, called "immune checkpoints", produces co-inhibitory or co-stimulatory signals. The immune checkpoints regulates the T-cell immunotolerance to protect the tissues from undesirable damages. Cancer cells may produce signals to inhibit T-cell action, through cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), programmed cell death-1 (PD-1) and its ligands (PDL-1) [37,40].
PD-1 is an inhibitory "immune checkpoint" expressed on the surface of T-cells, B-cells and NK-cells. When T-cells have been activated by their TCR, the cells express at the same time PD-1, which is a possibility to the attacked cell to escape from the immune reaction ( Figure 5). Cancer cells express the ligand PDL-1 on their surfaces, activating PD-1 of T-cells, escaping from the attack [37,40].
PD-L1 expression has been associated with large tumor size, high grade, high proliferation, estrogen receptor (ER)-negative status, and human epidermal growth factor receptor-2 (HER2)-positive status in breast cancer. Survival in breast cancer is inversely related to PD-1/PDL-1 levels. PDL-1 expression increases tumor

Simplified schematic illustration of PD-1/PDL-1 interactions in immune responses against cancer cell. Tumoral antigens (Ag) are presented via T-cell by major histocompatibility complex (MHC) of dendritic cells. T-cell recognize tumoral Ag via TCR (T-cell receptor). Interaction Ag-TCR induces an positive immune response against tumoral Ag. Though, there is a scape mechanism of cancer cell from the T-cell attack: interaction of programmed death cell ligands (PDL-1/2) expressed by cancer cell with PD-1 expressed by T-cell inhibit the T-cell action.
This scape mechanism of cancer cell mimics the regulation action to avoid immune responses of T-cell against self antigens. The principle of immune therapy is the inhibition of PD-1/PDL-1 (extracted from [40]). Advances in Molecular and Immunohistochemical Detection of Prognostic and Therapeutic… DOI: http://dx.doi.org /10.5772/intechopen.94462 aggressiveness, stimulating tumorigenesis, invasiveness and ability to escape from cytotoxic T CD8+ lymphocytes attacks [39,41]. The immunohistochemical evaluation of PDL-1 is shown in Figure 6.
Immune therapies with anti-CTLA-4 and anti-PD1/anti-PDL-1 agents have been promising for treating several cancers. In breast cancer, some researches reported positive results around 20% of breast tumors on treatment with these agents, mainly the "triple negative" and HER-2 subtypes, for their higher antigenicity. In general, breast cancer present lower immunogenicity than other cancers and breast cancer cells frequently create an immunosuppressor tumor microenvironment by signaling [37,43].
The presence of tumor infiltrating lymphocytes (TIL) in some breast cancers has been related to a favorable prognosis, especially in "triple negative" and HER-2 subtypes. TIL are formed mainly by T-cells CD3+/CD56 negative, which are either CD4+ or CD8+. A minority component of B-cells CD20+ and NK-cells may be present. The attraction of TIL by cancer cells have been related to their expression of some chemokines, like CXCL9 and CXCL13 [37,44].
In "triple negative" and HER-2 subtypes of breast cancer, the presence of TIL is related to a better response to neoadjuvant therapy, as well neoadjuvant treatment may modify the tumor microenvironment to attract TIL to tumor site. Furthermore, when the TIL are not attracted instead of neoadjuvance, it is indicative for bad prognosis [44].  [42]).

Advanced stage breast cancer: considerations under current approach and futures perspectives
Metastatic breast cancer is considered incurable nowadays with currently therapies. Therapy of metastatic disease aims to guarantee quality of life, palliation of symptoms and prolongation of the patient survival. Advanced stage disease is becoming increasingly chronic, controlled by sequencial therapies, with more personalized approach than the early stage breast cancer [8].
Systemic therapy is frequently the first choice of metastatic disease. Before the new therapeutic decision, it is necessary to consider the previous treatments. If possible, it is recommended to re-evaluate the histologic features and molecular subtype status of the metastatic lesion through a new biopsy, with new immunohistochemical study for hormonal receptor and HER-2 status. Some studies reported until 40% of discrepances of metastatic lesion histologic features and molecular subtype status versus primary tumor histologic and immunohistochemical aspects [45].
The metastatic disease therapeutic choices search for positive targets to hit more effectively the neoplastic cells. Thereby, expression of hormonal receptors by the metastatic lesion is elective for endocrine therapy. Endocrine drugs include tamoxifen, aromatase inhibitors, fulvestrant and progestins. The use of these drugs in metastasis with hormone receptor positive status have demonstrated increase of free of disease survival in several studies [8,45].
Furthermore, new generation of drugs which inhibit the cyclin dependant kinase (CDK) have been successful in prolongation of free of disease survival in luminal subtype HER-2 negative metastatic disease. CDK4/6 is a holoenzyme responsible for several extracellular signaling pathways to cell cycle transitions. CDK4/6 fosforilates and inactivates retinoblastoma tumor supressor protein (Rb). Extracellular signals regulate the expression of cyclins and CDK inhibitors, like p16 INK4a [46].
In human cancer, this circuit is dysregulated by either overexpression of cyclin D1, loss of p16 Ink4a , the mutation of CDK4 to an Ink4-refractory state, or the loss of Rb itself. The primary target of CDK4 is the Rb protein, though this holoenzyme either can phosphorylate factors involved in cell differentiation affecting their transcriptional activity, apoptotic factors affecting their activity and other factors that can directly affect mitochondrial function [8,46,47].
Therefore, CDK inhibitors act in tumor microenvironment, blocking Rb phosphorilation and leading to cell cycle exit. Moreover, CDK have kinase activity towards SPOP, an ubiquitin protein that interacts with PDL-1. CDK inhibitors lead to inhibition of SPOP phosphorilation with blockade of PDL-1 and stimulus to PD-1 expression by T-cells, attracting T-cell infiltration to the tumor. In this way, the combined use of CDK inhibitors and PDL-1/PD-1 inhibitors may be promising, requiring more future studies [46][47][48].
For the moment, hormonal receptors and HER-2 status are the few validated molecular targets of clinical importance on metastatic breast cancer approaching through chemotherapy and endocrine therapy. For HER-2 positive metastatic disease, anti-HER-2 treatment with trastuzumab is well established and is recommended as soon as possible. Immune therapy is not standardized for metastatic breast cancer, since metastatic breast disease is highly heterogeneous. Though, it is a promising therapy for the future, as well the target molecular therapies, which become more effective with discovery of novel pathways and mutations by new studies to be developed [8].
A resume of main biomarkers of clinicopathologic importance for breast cancer management is shown in Table 3 and a proposal of a algorithm for clinicopathologic evaluation of breast cancer is presented in

Conclusion and final considerations
In the 21st century, breast cancer classification and diagnosis advanced considerably from a purely morphologic/histologic approaching to a immune and molecular basis, with remarkable improvement of the correlation between classification and prediction of biological behavior and prognosis.
The adoption of a clinicopathologic classification based on molecular subtypes of breast cancer in the last decade has modified decisively the management of the disease in the way of molecular era, opening new ways to discovering of multiple targets for novel therapies.
Innovative concepts related to immune reactions related to human cancers, which have been unveiled in the recent years, particularly the immune checkpoints, have offered new treatment tools for several human cancers with promising results, although not still established for breast cancer.
In the molecular era of cancer, the integration of novel knowledges in a direction of more accurated diagnosis and prediction of prognosis to allow personalized therapies is the key to future human cancer management, including the breast cancer.
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.