Although significant progress has been made in the management of the hereditary cancer syndrome related to mutations of BRCA1, two fundamental and clinically relevant questions regarding BRCA1-related cancer syndrome remain unresolved: (1) What factors account forthe tissue specificity of the BRCA1-related cancerrisk? (2) How does a mutation orloss of BRCA1 lead to the basal-like phenotype of breast cancer? This review focuses on recent studies in BRCA1-related pathways that lead to specific characteristics of the hereditary cancer syndrome and discusses the current translational evidence for exploiting these pathways in new therapeutic strategies. Mounting evidence suggests that estrogen signaling and metabolism, oxidative stress, specific secondary mutations, and regulation of specific progenitor cells and transcriptional programs are critical in BRCA1-associated breast cancer. Strategies geared toward estrogen reduction may play a role in treatment and prevention. Therapies aimed at mitigating oxidative stress may be a strategy for risk reduction, while cancer-cellspecific sensitivity to oxidative stress may also be an opportunity for specific targeting. BRCA1-related transcriptional regulation and signaling provide a number of therapeutic targets, including the PI3-AKT and Notch pathways. Thus, significant opportunities exist in translational and clinical research for developing the treatment strategies forthe management of BRCA1-related breast cancer.

**Keywords:** BRCA1, basal-like breast cancer, DNA repair, estrogen, reactive oxygen species

© 2017 The Author(s). Licensee InTech. 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. © 2017 The Author(s). Licensee InTech. 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.

#### **1. Introduction**

In 1866, Pierre Paul Broca described the remarkable pedigree of his wife's family in his treatise *Traité des Tumeurs*: Of 19 women from five generations who lived to the age of 30 years, cancer developed in 14 women, including nine cases of breast cancer [1]. The French physician and surgeon observed: "This deadly predisposition, impossible to foretell, impossible to escape, inaccessible to surgery, and until now even inaccessible to internal or medical treatment, is an indication of a general state which precedes each local manifestation … in certain cases this predisposition transmits itself by heredity through several generations" [1]. Progress was slow for the next 100 years. Further identification of family pedigrees suggested the hereditary passage of breast cancer, but this remained controversial [2]. With the second half of the twentieth century came the recognition of a familial breast cancer syndrome that in some cases could be associated with an autosomal dominant allele encoding a tumor suppressor gene [3]. In 1990, linkage analysis mapped the putative allele to chromosome 17q21, and the gene, BRCA1, was finally cloned in 1994 [4, 5]. The identification of BRCA2 followed a similar trajectory, with its localization to 13q12-13 in 1994 and cloning the following year [6, 7]. In addition to BRCA1/2, a number of genes have been implicated in familial breast cancer with varying degrees of conferred risk that are generally inversely related to population allele frequency; BRCA1, for example, has rare mutations with high penetrance [8]. Since that time, significant advances have been made in understanding the risks and mechanisms of BRCA1/2 related carcinogenesis. For patients with BRCA1/2 mutations, estimates of lifetime cancer risk widely vary due to different studies of cohorts and varying penetrance attributed to different mutation-related phenotypes, family/genetic history, and environmental exposures. Cumulative risk by 70 years of age for patients with germline BRCA1 mutations ranges between 46% and 87% for breast cancer and between 27% and 63% for ovarian cancer, with the low-risk and general population studies falling in the lower range of the estimate and high-risk families in the upper range. For BRCA2 mutations, the risks are 31–56% and up to 11%, respectively [9].

A number of therapeutic strategies have been developed to manage hereditary breast cancer. The National Comprehensive Cancer Network evidence-based guidelines provide an algorithm for the management of hereditary breast cancer, including identification of high-risk individuals and families, genetic testing, cancer screening, risk mitigation through prophylactic salpingo-oophorectomy and possible mastectomy, locoregional and systemic treatment, and ongoing surveillance [11]. Newer therapies and combination strategies are being designed to target more specific features of the cancer genotype and phenotype. The use of PARP inhibitors in the model of synthetic lethality to exploit deficiency in homologous repair in BRCA1/2 deficient cells is one such example [12–15]. This targets one aspect of BRCA1/2 function, but the development of further strategies is desirable, especially as BRCA1-related cancer and cancer risk have a more complicated etiology than defective homologous repair alone. The BRCA1 cancer syndrome may be related to the myriad cellular processes in which BRCA1 is involved, including recognition and repair of genomic damage, regulation of chromosome sorting and mitosis, control of cell-cycle checkpoints, protein ubiquitination, cell signaling, and transcriptional regulation [16–20]. Further, abrogation of BRCA1 function can be the result of a multitude of lesions and processes with variable penetrance and relative hypomorphism, including mutations resulting in a premature stop codon with or without mRNA transcription and expression of a truncated protein, mutations resulting in loss of the function of particular functional domains, intronic mutations resulting in splice variants, large rearrangements, variation in mRNA splicing, methylation and silencing of the gene, and regulation by microRNA [21–23]. Although most BRCA1 mutant mRNAs have shortened halflife, some BRCA1 mutant proteins are translated [24]. However, characterizing cellular localization and function of these proteins is difficult. Staining for intracellular BRCA1 in mutants is inconsistent, as patterns vary with different antibodies, fixation methods, and methods of exposing epitopes, which may lead to a lack of correlation between BRCA1 staining and qPCR levels [25, 26]. Despite nearly two decades of genetic testing, there remain a significant number of variants of uncertain significance. Even when a mutation is established as pathogenic, targeted therapies against DNA repair may not always be effective, as preclinical data suggest in the case of at least one common pathogenic mutation, C61G [27].

Out of this complicated picture, two fundamental questions arise regarding specific characteristics of the BRCA1-related cancer syndrome, both with the potential to guide cancer prevention and therapy: (1) What accounts for the tissue specificity of the BRCA1-related cancer risk? (2) How does mutation or loss of BRCA1 lead to the basal-like phenotype of breast cancer, as opposed to the luminal phenotype of BRCA2 mutations? We searched PubMed for English-language studies and reviews related to BRCA1 function, estrogen metabolism, oxidative stress, basal-like breast cancer, and BRCA1-related therapy. Reference lists of selected articles were searched to track the provenience of key ideas and findings.

### **2. BRCA1/2 and carcinogenesis**

**1. Introduction**

328 Breast Cancer - From Biology to Medicine

and prostate cancer [10].

In 1866, Pierre Paul Broca described the remarkable pedigree of his wife's family in his treatise *Traité des Tumeurs*: Of 19 women from five generations who lived to the age of 30 years, cancer developed in 14 women, including nine cases of breast cancer [1]. The French physician and surgeon observed: "This deadly predisposition, impossible to foretell, impossible to escape, inaccessible to surgery, and until now even inaccessible to internal or medical treatment, is an indication of a general state which precedes each local manifestation … in certain cases this predisposition transmits itself by heredity through several generations" [1]. Progress was slow for the next 100 years. Further identification of family pedigrees suggested the hereditary passage of breast cancer, but this remained controversial [2]. With the second half of the twentieth century came the recognition of a familial breast cancer syndrome that in some cases could be associated with an autosomal dominant allele encoding a tumor suppressor gene [3]. In 1990, linkage analysis mapped the putative allele to chromosome 17q21, and the gene, BRCA1, was finally cloned in 1994 [4, 5]. The identification of BRCA2 followed a similar trajectory, with its localization to 13q12-13 in 1994 and cloning the following year [6, 7]. In addition to BRCA1/2, a number of genes have been implicated in familial breast cancer with varying degrees of conferred risk that are generally inversely related to population allele frequency; BRCA1, for example, has rare mutations with high penetrance [8]. Since that time, significant advances have been made in understanding the risks and mechanisms of BRCA1/2 related carcinogenesis. For patients with BRCA1/2 mutations, estimates of lifetime cancer risk widely vary due to different studies of cohorts and varying penetrance attributed to different mutation-related phenotypes, family/genetic history, and environmental exposures. Cumulative risk by 70 years of age for patients with germline BRCA1 mutations ranges between 46% and 87% for breast cancer and between 27% and 63% for ovarian cancer, with the low-risk and general population studies falling in the lower range of the estimate and high-risk families in the upper range. For BRCA2 mutations, the risks are 31–56% and up to 11%, respectively [9].  The National Comprehensive Cancer Network evidence-based guidelines provide an algorithm for the management of hereditary breast cancer, including identification of high-risk individuals and families, genetic testing, cancer screening, risk mitigation through prophylactic salpingo-oophorectomy and possible mastectomy, locoregional and systemic treatment, and ongoing surveillance [11]. Newer therapies and combination strategies are being designed to target more specific features of the cancer genotype and phenotype. The use of PARP inhibitors in the model of synthetic lethality to exploit deficiency in homologous repair in BRCA1/2 deficient cells is one such example [12–15]. This targets one aspect of BRCA1/2 function, but the development of further strategies is desirable, especially as BRCA1-related cancer and cancer risk have a more complicated etiology than defective homologous repair alone. The BRCA1 cancer syndrome may be related to the myriad cellular processes in which BRCA1 is involved, including recognition and repair of genomic damage, regulation of The best known and perhaps dominant roles for the BRCA proteins in tumor suppression lie in the maintenance of chromosomal stability, a role played in nearly all tissues [28]. Both BRCA1 and BRCA2 are essential for homologous repair, a high-fidelity repair mechanism for double-stranded breaks and daughter strand gaps, lesions that can arise from DNA damage and at stalled replication forks [29, 30]. Lack of BRCA proteins results in these lesions being shunted into error-prone repair pathways resulting in chromosomal rearrangements and deletions [30–32]. Both BRCA1/2 have additional roles in chromosomal stability: BRCA1 functions in the recognition of DNA damage and the recruitment and assembly of protein complexes for repair of lesions, and BRCA2 stabilizes stalled replication forks to allow for repair rather than degradation and prevents spontaneous hyperrecombination [28, 33, 34]. BRCA1 also heterodimerizes with BARD1 at the N-terminal ring domain, conferring E3 ubiquitin ligase activity and regulating mitotic spindle assembly; loss of this interaction also results in loss of tumor suppressor activity [27, 35]. Dysregulation of the mitotic spindle assembly as well as centrosome amplification, along with failure of the G2-M checkpoint, leads to defects in chromosome segregation, abnormal division, and aneuploidy [35, 36]. BRCA2 may also have a role in cell-cycle checkpoint control [37].

Maintenance of chromosomal stability alone cannot explain comprehensively the related cancer syndrome beyond an increased risk of carcinogenesis. Other factors are needed to account for the tissue tropism of BRCA1/2-related cancers as well as the particular phenotype of BRCA1-related cancer: basal subtype cancer developing specifically in the epithelium of the breast and papillary serous cancer developing most likely in the fimbria of the fallopian tube —cancers that have similar mutational profiles and likely similar early driving events in carcinogenesis [38]. Several explanations may account for the tissue specificity of the carcinogenic potential. First, breast and fallopian tube cells are subject to a unique exposure resulting in accumulating mutations and genomic damage, possibly related to the genotoxic effects of estrogen metabolism and the generation of reactive oxygen species (ROS), or related to abrogation of normal cell-cycle control in tissues periodically undergoing multiple cycles of rapid proliferation. These tissues may provide an environment that is permissive for, or even driving, cell survival and proliferation despite mounting genomic damage. Further, functions of BRCA1 unrelated to genomic stability may contribute both to the tissue-specific risk as well as the particular phenotype including transcription-related roles in the regulation of mammary progenitor cells and moderation of the proliferative effects of estrogen signaling.

#### **2.1. BRCA1 cancer tissue specificity: estrogen and oxidative stress**

Maintenance of the genome alone cannot explain the tissue-specific nature of BRCA1-related cancer risk. It is well documented that cumulative estradiol exposure is linked to lifetime risk of the development of breast cancer [39]. Estrogen-linked carcinogenesis could be related to the transcriptional program of estrogen signaling, which promotes cell proliferation, or to the toxic side effects of estrogen metabolism. BRCA1 interacts with the classical estrogen signaling pathway in combination with BARD1 by repressing ERα-related transcription through ubiquitination, a function lost with deleterious mutations of the BRCA1 RING domain [40, 41]. However, estrogen signaling is not restricted to the nuclear receptors ERα and ERβ and likely plays a role in estrogen receptor negative cancers. Recent studies have shown an alternative mechanism of BRCA1 cell survival based on nonclassical binding of estrogen to cytoplasmic and membrane-associated proteins with downstream effects preventing damage from oxidative stress [42]. Gorrini et al. showed that both oxidative stress and estrogen induced the expression of NRF2, a master regulator of antioxidant capacities, through the PI3K-AKT pathway, and that NRF2 induced by estrogen was crucial for cell survival. They also showed that apoptosis may be prevented in BRCA1 knockdown mammary epithelial cells with exposure to estrogen [43, 44]. This is consistent with clinical observations that reduction in estrogen load reduces risk of cancer in women carrying a BRCA mutation, even if BRCA1 related cells are estrogen receptor negative [45].

BRCA1 mutants may be particularly sensitive to estrogen metabolites, and the early risk of developing cancer reflects the rapid, uncorrected accumulation of genotoxic damage from exposure to both estrogen metabolites and reactive oxygen species produced by oxidative metabolism of estrogen through the catechol pathway, a topic reviewed by Yager and Davidson [39]. Metabolism of estrogen by cytochrome P-450 enzymes, including some tissue-specific enzymes, leads to the formation of estrogen-3,4-quinone, which can form stable DNA adducts and depurinating DNA adducts resulting in mutagenesis. Reduction of oxidized estrogen metabolites leads to reactive oxygen species, which may further damage DNA, proteins, and lipids [39]. Recently, Santen et al. have shown in ER knockout mice the dose-dependent accumulation of toxic estrogen metabolites and concordant rates of tumor formation along with mitigation by estrogen reduction via oophorectomy or aromatase inhibitor treatment [46]. Further, Savage et al. showed that treatment with the estrogen metabolites, 2-hydroxyestradiol and 4-hydroxyestradiol, resulted in double-strand breaks, produced primarily during S-phase, and that BRCA1 deficiency, including both heterozygous and homozygous mutants, led to increased double-strand breaks and loss of efficient repair [47]. Further, wild-type BRCA1 represses the expression of estrogen metabolizing genes, resulting in decreased damage to DNA [47].

to defects in chromosome segregation, abnormal division, and aneuploidy [35, 36]. BRCA2

Maintenance of chromosomal stability alone cannot explain comprehensively the related cancer syndrome beyond an increased risk of carcinogenesis. Other factors are needed to account for the tissue tropism of BRCA1/2-related cancers as well as the particular phenotype of BRCA1-related cancer: basal subtype cancer developing specifically in the epithelium of the breast and papillary serous cancer developing most likely in the fimbria of the fallopian tube —cancers that have similar mutational profiles and likely similar early driving events in carcinogenesis [38]. Several explanations may account for the tissue specificity of the carcinogenic potential. First, breast and fallopian tube cells are subject to a unique exposure resulting in accumulating mutations and genomic damage, possibly related to the genotoxic effects of estrogen metabolism and the generation of reactive oxygen species (ROS), or related to abrogation of normal cell-cycle control in tissues periodically undergoing multiple cycles of rapid proliferation. These tissues may provide an environment that is permissive for, or even driving, cell survival and proliferation despite mounting genomic damage. Further, functions of BRCA1 unrelated to genomic stability may contribute both to the tissue-specific risk as well as the particular phenotype including transcription-related roles in the regulation of mammary

progenitor cells and moderation of the proliferative effects of estrogen signaling.

Maintenance of the genome alone cannot explain the tissue-specific nature of BRCA1-related cancer risk. It is well documented that cumulative estradiol exposure is linked to lifetime risk of the development of breast cancer [39]. Estrogen-linked carcinogenesis could be related to the transcriptional program of estrogen signaling, which promotes cell proliferation, or to the toxic side effects of estrogen metabolism. BRCA1 interacts with the classical estrogen signaling pathway in combination with BARD1 by repressing ERα-related transcription through ubiquitination, a function lost with deleterious mutations of the BRCA1 RING domain [40, 41]. However, estrogen signaling is not restricted to the nuclear receptors ERα and ERβ and likely plays a role in estrogen receptor negative cancers. Recent studies have shown an alternative mechanism of BRCA1 cell survival based on nonclassical binding of estrogen to cytoplasmic and membrane-associated proteins with downstream effects preventing damage from oxidative stress [42]. Gorrini et al. showed that both oxidative stress and estrogen induced the expression of NRF2, a master regulator of antioxidant capacities, through the PI3K-AKT pathway, and that NRF2 induced by estrogen was crucial for cell survival. They also showed that apoptosis may be prevented in BRCA1 knockdown mammary epithelial cells with exposure to estrogen [43, 44]. This is consistent with clinical observations that reduction in estrogen load reduces risk of cancer in women carrying a BRCA mutation, even if BRCA1-

BRCA1 mutants may be particularly sensitive to estrogen metabolites, and the early risk of developing cancer reflects the rapid, uncorrected accumulation of genotoxic damage from exposure to both estrogen metabolites and reactive oxygen species produced by oxidative metabolism of estrogen through the catechol pathway, a topic reviewed by Yager and David-

**2.1. BRCA1 cancer tissue specificity: estrogen and oxidative stress**

related cells are estrogen receptor negative [45].

may also have a role in cell-cycle checkpoint control [37].

330 Breast Cancer - From Biology to Medicine

Reactive oxygen species (ROS) are produced during normal aerobic metabolism and multiple other cellular processes. ROS and the redox state of a cell are also essential components in cell signaling and homeostasis. Imbalance of pro- and antioxidant factors, whether from endogenous sources or exogenous sources (e.g., UV radiation and tobacco), results in oxidative damage to nucleic acids, amino acids, and fatty acids, and contributes to a number of disease processes. Among the most common DNA lesions resulting from oxidative stress is 8 oxoguanine, which results in a mutagenic template during DNA replication resulting in base pair substitutions and stalling of RNA polymerase II at the site of the lesion with inhibition of nucleotide excision repair [48, 49]. Bae et al. showed that BRCA1 has a role in the response to oxidative stress by upregulating expression of antioxidant genes and enhancing the activity of NRF2 [50]. Further, BRCA1 maintains balance of the cellular redox state, making cells more resistant to exogenous oxidative stress. BRCA1 overexpression and deficiency result in increased and decreased resistance to exogenous oxidative stress, respectively [50]. Besides activating cellular defenses to oxidative stress, BRCA1/2 also mediate repair of DNA damage resulting from oxidative stress. Le Page et al. showed that BRCA1- and BRCA2-deficient cells are unable to repair 8-oxoG lesions and that reconstitution of wild-type BRCA proteins leads to recovery of the transcription-coupled repair mechanism [49]. These studies suggest that BRCA1 tumor suppression involves mitigating the damage of oxidative stress before it is required to repair the resulting DNA lesions.

#### **2.2. BRCA1 cancer phenotype: progenitor cells and transcriptional regulation**

Tumors arising in the setting of a germline BRCA1 mutation share common features from the level of genomic alterations, gene expression, histologic phenotype, clinical behavior and prognosis, and response to therapy. Histologically, they are high grade with high mitotic index, pushing tumor margins, central necrosis, and a lymphocytic infiltrate [51]. A subset of sporadic tumors, often demonstrating a relative decrease in BRCA expression through mechanisms other than germline loss, and arising in the same tissues as germline mutants, appears to share this constellation of traits [52]. Turner et al. coined the term BRCAness to identify "the phenotypes that some sporadic tumors share with familial-BRCA cancers" [52]. This is in contrast to BRCA2-related breast cancer, which has a significantly different gene expression profile, and which is more typically lower grade, more differentiated, appearing later in life, and of the luminal/ER-positive subtype [51, 53]. Interestingly, the relative risk profiles for BRCA1 and BRCA2 mutations are also different, further suggesting different etiologic mechanisms. Compared to the aged-matched general population, the relative risk for BRCA1 mutation carriers is greatest in the young population and approaches the population risk in the later decades of life; the relative risk for BRCA2 remains constantly elevated over the population risk throughout the patient's lifetime [54].

BRCAness may be a feature related to the progenitor cell of origin from which these cancers arise. Foulkes hypothesized that BRCA1 acting as a regulator of mammary stem cell function may drive the phenotype of BRCA1-related cancers [55]. In this model, immortal mammary stem cells absent a BRCA1 signal would maintain a relatively undifferentiated, proliferative phenotype that would require very few additional genomic "hits" in order to become malignant; genomic instability conferred by loss of BRCA1-mediated DNA repair functions would account for the proclivity for malignant derangement and the early age of presentation. The general model is appealing, although it appears more likely that BRCA1-associated cancer arises from luminal epithelial progenitors, not mammary stem cells [56].

The mammary epithelium can be sorted into subgroups representing different stages of the differentiation from multipotent mammary stem cell to mature luminal epithelium, which requires BRCA1 for proper development [57]. Depletion of BRCA1 results in failure of mammary cells to differentiate and form acini in culture, but increases cell proliferation [58]. Liu et al. showed that BRCA1 knockdown increases stem/progenitor cell population while preventing mammosphere formation [59]. Furthermore, in human mammary tissue, BRCA1 heterozygotes showed lobules comprised of ALDH1 (stem cell marker) positive cells with minimal ER expression and evidence of BRCA1 loss of heterozygosity. These lobules occurred in normal tissue, showing that mammary progenitor cells can survive without BRCA1 expression and create atypical, nonmalignant lobules. This suggests that the tissue tropism of BRCA1-related tumors is due to a permissive environment, possibly due to release of paracrine factors from luminal epithelium, for the survival of BRCA1 negative cells [60].

Lim et al. [62] demonstrated that normal mammary cells sorted by basal and epithelial markers showed varied potency and clonogenic activity *in vivo* corresponding to bipotent progenitor, committed luminal progenitor, and mature luminal cells. These may represent the cells of origin of the different subtypes of mammary epithelial tumors that can be segregated by gene expression [53, 61]. Comparison of gene expression profiles between normal mammary subpopulations of mammary stem cells, luminal progenitor cells, and mature luminal cells showed significant associations with, respectively, claudin-low, basal, and luminal A and B cancer cell populations [62]. Further analysis of these subtypes reveals few somatic mutations common to all breast cancers, but within the well-defined subtypes, genetic and epigenetic changes give rise to their common phenotypes [38]. These tumor subtypes can also be segregated by clinical behavior, prognosis, and response to treatment [63].

Gene-expression profiles of BRCA1-associated tumors correlated most closely with luminal progenitor cells and loss of BRCA1 in a mouse luminal breast cancer model leads to epithelialmesenchymal transition (EMT), dedifferentiation, and basal tumor development [62, 64, 65]. BRCA1 transcriptionally regulates a number of genes associated with basal-like breast cancer, including Notch ligands and receptors, with loss of BRCA1 associated with decreased luminal differentiation and ER-α signaling and also with increased basal-like and proliferation markers [66]. Increased Notch signaling due to BRCA1 loss may contribute to the basal-like phenotype as well as suppression of apoptosis [67]. Wild-type BRCA1 represses expression of a number of genes associated with basal-like and BRCA1-related cancers, including FOXC1, *p*-Cadherin, and CK5 and 17 [68, 69]. The luminal progenitor, as the cell of origin for BRCA1-related cancer, is consistent with an important role for ROS as mammary stem cells and multipotent progenitor cells have lower concentrations of ROS than more mature progenitor cells [70].

Around 80–90% of BRCA1 tumors are basal, as opposed to 10–15% of all tumors, although may also sort with the claudin-low subtype [38, 71, 72]. Conversely, around 20% of basal tumors show germline or somatic BRCA1/2 mutation [38]. BRCA1-related cancers exhibit common mutational profiles. A total of 81–89% of BRCA1 tumors, both ER+ and ER−, have a loss of heterozygosity of the wild-type allele, which is correlated with higher grade and increased proliferation [73]. For cells lacking functional BRCA1, cell survival is generally dependent on secondary mutations. BRCA1 tumors commonly show mutations of PTEN and TP53 allowing cell proliferation to continue in spite of mounting genomic irregularities. These mutations appear to follow a general evolutionary pattern preceding the loss of the WT BRCA allele [74]. This is significantly different than tumorigenesis in luminal cancers, with rare PTEN mutations and late loss of TP53. Even without loss of wild-type allele, heterozygotes display altered gene expression, including in genes related to cell differentiation and proliferation [75].

### **3. Developments in targeted therapy**

contrast to BRCA2-related breast cancer, which has a significantly different gene expression profile, and which is more typically lower grade, more differentiated, appearing later in life, and of the luminal/ER-positive subtype [51, 53]. Interestingly, the relative risk profiles for BRCA1 and BRCA2 mutations are also different, further suggesting different etiologic mechanisms. Compared to the aged-matched general population, the relative risk for BRCA1 mutation carriers is greatest in the young population and approaches the population risk in the later decades of life; the relative risk for BRCA2 remains constantly elevated over the

BRCAness may be a feature related to the progenitor cell of origin from which these cancers arise. Foulkes hypothesized that BRCA1 acting as a regulator of mammary stem cell function may drive the phenotype of BRCA1-related cancers [55]. In this model, immortal mammary stem cells absent a BRCA1 signal would maintain a relatively undifferentiated, proliferative phenotype that would require very few additional genomic "hits" in order to become malignant; genomic instability conferred by loss of BRCA1-mediated DNA repair functions would account for the proclivity for malignant derangement and the early age of presentation. The general model is appealing, although it appears more likely that BRCA1-associated cancer

The mammary epithelium can be sorted into subgroups representing different stages of the differentiation from multipotent mammary stem cell to mature luminal epithelium, which requires BRCA1 for proper development [57]. Depletion of BRCA1 results in failure of mammary cells to differentiate and form acini in culture, but increases cell proliferation [58]. Liu et al. showed that BRCA1 knockdown increases stem/progenitor cell population while preventing mammosphere formation [59]. Furthermore, in human mammary tissue, BRCA1 heterozygotes showed lobules comprised of ALDH1 (stem cell marker) positive cells with minimal ER expression and evidence of BRCA1 loss of heterozygosity. These lobules occurred in normal tissue, showing that mammary progenitor cells can survive without BRCA1 expression and create atypical, nonmalignant lobules. This suggests that the tissue tropism of BRCA1-related tumors is due to a permissive environment, possibly due to release of paracrine

Lim et al. [62] demonstrated that normal mammary cells sorted by basal and epithelial markers showed varied potency and clonogenic activity *in vivo* corresponding to bipotent progenitor, committed luminal progenitor, and mature luminal cells. These may represent the cells of origin of the different subtypes of mammary epithelial tumors that can be segregated by gene expression [53, 61]. Comparison of gene expression profiles between normal mammary subpopulations of mammary stem cells, luminal progenitor cells, and mature luminal cells showed significant associations with, respectively, claudin-low, basal, and luminal A and B cancer cell populations [62]. Further analysis of these subtypes reveals few somatic mutations common to all breast cancers, but within the well-defined subtypes, genetic and epigenetic changes give rise to their common phenotypes [38]. These tumor subtypes can also be

Gene-expression profiles of BRCA1-associated tumors correlated most closely with luminal progenitor cells and loss of BRCA1 in a mouse luminal breast cancer model leads to epithelial-

arises from luminal epithelial progenitors, not mammary stem cells [56].

factors from luminal epithelium, for the survival of BRCA1 negative cells [60].

segregated by clinical behavior, prognosis, and response to treatment [63].

population risk throughout the patient's lifetime [54].

332 Breast Cancer - From Biology to Medicine

Therapy for BRCA1/2 related tumors involves surgery, radiation, and systemic chemotherapy and endocrine therapy. Specific treatment for cancer developing in the setting of BRCA1/2 has targeted deficient DNA repair. Platinum-based chemotherapy creates intra- and interstrand DNA crosslinks resulting in double-stranded breaks. In the absence of homologous repair, the accumulation of genomic damage results in cell death. Pegylated liposomal doxorubicin has also been shown to have a survival advantage [76].

PARP inhibitors have been used to exploit the DNA repair defect [13]. PARP-1 binds to DNA strand breaks and signals DNA damage by hydrolyzing NAD+ to form poly(ADP-ribosyl) tails on histones and itself, resulting in the recruitment of the protein machinery for repair [77]. PARP inhibitors include a nicotinamide moiety that competes with NAD+, inhibiting the enzymatic function of PARP, and trapping the PARP enzyme at the site of DNA damage, preventing repair [78]. Loss of PARP-mediated regulation and repair of single strand breaks leads to stalled replication forks, and double strand breaks develop, which leads to cell death in a process referred to as synthetic lethality. Loss of BRCA1 prevents homologous repair from occurring; loss of PARP function results in loss of regulation of nonhomologous end joining, which leads to error-prone repair, genomic instability, and cell death [79]. A number of clinical trials assessing the efficacy of PARP inhibitors in BRCA-mutated breast cancer are currently underway, in metastatic disease as well as in the adjuvant and neoadjuvant setting (http:// www.cancer.gov/about-cancer/treatment/clinical-trials).

However, not all BRCA1-related tumors are sensitive to DNA damaging agents and PARP inhibitors. BRCA1 deleterious mutations in the RING finger domain lose tumor suppressor function related to loss of interaction with PALB but retain some homologous repair activity, rendering them less responsive to PARP inhibitors and platinum chemotherapy [27]. There is also evidence that BRCA1/2-associated tumors gain resistance to platinum and PARP inhibitor therapy through mutations resulting in reversion to the wild-type sequence or other restoration of the open reading frame [80]. Lord and Ashworth also review preclinical data suggesting that loss of 53BP1 or the related RAP1-interacting factor 1 (RIF1), proteins involved in nonhomologous end joining, leads to reduced genomic damage from PARPi-induced nonhomologous end joining and at least partial restoration of homologous repair and survival for BRCA1 deficient cells. However, clinical data supporting this mechanism are lacking [80].

Further specific treatment for BRCA1 cancer risk includes targeting estrogen production and signaling and prophylactic surgery to eliminate or reduce the number of potential tumorigenic cells. Bilateral salpingo-oophorectomy has been shown to significantly reduce the risk of breast cancer incidence by about 50% and breast cancer mortality by 90% [45, 81]. The use of tamoxifen, independent of estrogen receptor status, reduced the risk of contralateral breast cancer in BRCA1 mutation carriers with HR 0.38 (95% CI, 0.27–0.55) in a pooled prospective/retrospective cohort [82]. Specific therapy to prevent formation of ROS or toxic estrogen metabolites without endocrine ablation or blockade present a possible target that would modify cancer risk without the possible side effects and complications of surgery and iatrogenic menopause, including loss of fertility. Alternatively, strategies that exploit increased oxidative stress in tumor cells may provide strategy for targeted therapy. In normal cells, ROS are produced at low concentrations and can be effectively neutralized by the antioxidant system of the cells. In contrast, cancer cells produce elevated levels of ROS due to increased metabolic activity, resulting in a state of chronic oxidative stress. As noted above, BRCA1-mutant cells have a dysregulated response and increased sensitivity to oxidative stress as well as a decreased ability to repair DNA lesions resulting from ROS. As such, induction of ROS-mediated damage in cancer cells by proper pharmacological agents that either promote ROS generation beyond the cellular antioxidative capacity or disable the cellular antioxidant system have been considered as a "radical" therapeutic strategy to preferentially kill cancer cells [83]. Elesclomol, a small molecule that increases ROS production in mitochondria and induces apoptosis, has shown *in vitro* potential for treating breast cancer cells with defective DNA repair [84, 85].

The PI3K-AKT pathway is another target for therapy. PI3K is involved in both oxidative stress and escape mechanisms of DNA repair from PARP inhibitors. Combination treatment with PI3K and PARP inhibitors showed significant efficacy in inhibiting tumor cell growth *in vitro* and reducing tumor volume in mouse models [86]. PI3-AKT also functions downstream of Notch, a critical cancer stem cell regulator associated with basal-like breast cancer, in suppression of apoptosis [87, 88]. Although targeting the Notch pathway alone is not sufficient to reduce proliferation or cause cell death, combination with inhibition of the EGFR pathway or AKT pathway results in enhanced cell death [88]. These results suggest that combination therapies targeting signaling pathways implicated in basal-like breast cancer or BRCA1-regulated cell function would provide new avenues for combating BRCA1-related breast cancer.

### **4. Conclusion**

trials assessing the efficacy of PARP inhibitors in BRCA-mutated breast cancer are currently underway, in metastatic disease as well as in the adjuvant and neoadjuvant setting (http://

However, not all BRCA1-related tumors are sensitive to DNA damaging agents and PARP inhibitors. BRCA1 deleterious mutations in the RING finger domain lose tumor suppressor function related to loss of interaction with PALB but retain some homologous repair activity, rendering them less responsive to PARP inhibitors and platinum chemotherapy [27]. There is also evidence that BRCA1/2-associated tumors gain resistance to platinum and PARP inhibitor therapy through mutations resulting in reversion to the wild-type sequence or other restoration of the open reading frame [80]. Lord and Ashworth also review preclinical data suggesting that loss of 53BP1 or the related RAP1-interacting factor 1 (RIF1), proteins involved in nonhomologous end joining, leads to reduced genomic damage from PARPi-induced nonhomologous end joining and at least partial restoration of homologous repair and survival for BRCA1-

deficient cells. However, clinical data supporting this mechanism are lacking [80].

Further specific treatment for BRCA1 cancer risk includes targeting estrogen production and signaling and prophylactic surgery to eliminate or reduce the number of potential tumorigenic cells. Bilateral salpingo-oophorectomy has been shown to significantly reduce the risk of breast cancer incidence by about 50% and breast cancer mortality by 90% [45, 81]. The use of tamoxifen, independent of estrogen receptor status, reduced the risk of contralateral breast cancer in BRCA1 mutation carriers with HR 0.38 (95% CI, 0.27–0.55) in a pooled prospective/retrospective cohort [82]. Specific therapy to prevent formation of ROS or toxic estrogen metabolites without endocrine ablation or blockade present a possible target that would modify cancer risk without the possible side effects and complications of surgery and iatrogenic menopause, including loss of fertility. Alternatively, strategies that exploit increased oxidative stress in tumor cells may provide strategy for targeted therapy. In normal cells, ROS are produced at low concentrations and can be effectively neutralized by the antioxidant system of the cells. In contrast, cancer cells produce elevated levels of ROS due to increased metabolic activity, resulting in a state of chronic oxidative stress. As noted above, BRCA1-mutant cells have a dysregulated response and increased sensitivity to oxidative stress as well as a decreased ability to repair DNA lesions resulting from ROS. As such, induction of ROS-mediated damage in cancer cells by proper pharmacological agents that either promote ROS generation beyond the cellular antioxidative capacity or disable the cellular antioxidant system have been considered as a "radical" therapeutic strategy to preferentially kill cancer cells [83]. Elesclomol, a small molecule that increases ROS production in mitochondria and induces apoptosis, has shown *in vitro* potential for treating breast cancer cells with defective DNA repair [84, 85].

The PI3K-AKT pathway is another target for therapy. PI3K is involved in both oxidative stress and escape mechanisms of DNA repair from PARP inhibitors. Combination treatment with PI3K and PARP inhibitors showed significant efficacy in inhibiting tumor cell growth *in vitro* and reducing tumor volume in mouse models [86]. PI3-AKT also functions downstream of Notch, a critical cancer stem cell regulator associated with basal-like breast cancer, in suppression of apoptosis [87, 88]. Although targeting the Notch pathway alone is not sufficient to reduce proliferation or cause cell death, combination with inhibition of the EGFR

www.cancer.gov/about-cancer/treatment/clinical-trials).

334 Breast Cancer - From Biology to Medicine

A great volume of knowledge regarding the molecular functions of BRCA1 and BRCA2 has developed in the last 20 years since the cloning of the genes. Despite some challenging issues in understanding BRCA1-mutant breast cancer development, there is great potential for advances in tying elucidated molecular pathways in cell and animal models to the clinical and epidemiological presentation of BRCA1/2-related breast cancer ( **Table 1**). Such translational advances may be exploited not only to advance the treatment of breast cancer but also to diminish the risk described by Pierre Paul Broca—inaccessible to treat, impossible to escape so long ago.

**Table 1.** Challenges and opportunities in BRCA1-related breast cancer research and treatment.

### **Acknowledgements**

We apologize to many scientists and clinicians whose work was not referenced due to space limitations. This work was supported by National Institutes of Health (CA151610), the Avon Foundation (02-2014-063), Eleanor and Glenn Padnick Discovery Fund in Cellular Therapy, and David Salomon Translational Breast Cancer Research Fund to XC, and the Fashion Footwear Charitable Foundation of New York, Inc., the Entertainment Industry Foundation, Associates for Breast and Prostate Cancer Studies, the Margie and Robert E. Petersen Foundation, Linda and Jim Lippman Research Fund to AG. The funders/sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. The authors declare no conflicts of interest.

### **Author details**

Jeffrey Johnson1 , William Audeh2,3, Hisashi Tanaka1,2, Farin Amersi1,2, Armando E. Giuliano1,2 and Xiaojiang Cui1,2\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA

2 Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA

3 Division of Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA

#### **References**

[8] Gracia-Aznarez FJ, Fernandez V, Pita G, Peterlongo P, Dominguez O, de la Hoya M, et al. Whole exome sequencing suggests much of non-BRCA1/BRCA2 familial breast cancer is due to moderate and low penetrance susceptibility alleles. PLoS One. 2013;8(2):e55681.

dation, Linda and Jim Lippman Research Fund to AG. The funders/sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the

, William Audeh2,3, Hisashi Tanaka1,2, Farin Amersi1,2,

1 Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA

2 Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center,

3 Division of Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, CA, USA

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**Author details**

336 Breast Cancer - From Biology to Medicine

Jeffrey Johnson1

Los Angeles, CA, USA

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#### **Molecular Fingerprints and Biomarkers of Breast Cancer Molecular Fingerprints and Biomarkers of Breast Cancer**

Hala Fawzy Mohamed Kamel, Hiba Saeed Bagader Al-Amodi and Hanan Mohamed AbdElmoneim Hala Fawzy Mohamed Kamel, Hiba Saeed Bagader Al-Amodi and Hanan Mohamed AbdElmoneim

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66899

#### **Abstract**

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342 Breast Cancer - From Biology to Medicine

2319–27.

Substantial progress has been made over the past three decades in understanding breast cancer (BC) molecular biology, genomics, and targeted therapy. The recent comprehensive molecular and pathological diversity observed in BC patients indicates that BC is not a homogeneous disease; It may be appropriately defined as a myriad of diseases. The explosion of molecular information in the past 10 years has led to a better understanding of the biologic diversity of breast cancers (BCs), and clues to the different etiologic pathways to BC development. It will be useful to study the epigenetics of BC cells and define the mechanisms of both genetic and epigenetic driving alterations beside the mutations. Identifying the oncogenes and tumor suppressor genes is the purpose cancer diagnostics and therapeutics. Oncogenes as well as novel ones involved in the significantly altered regions would enable researchers to identify new causes and molecular pathways that may be targeted at BC treatment. Our main goal is to provide comprehensive understanding of underlying molecular mechanisms and hallmarks of BC, focusing on the identification of fingerprints and novel molecular targets that will greatly improve the cancer predictive, prognostic, and diagnostic biomarkers and, in addition, the possible targets for novel therapies.

**Keywords:** breast cancer, carcinogenesis, molecular markers, omics, personalized medicine

### **1. Introduction**

Cancers, including breast cancer, are generally thought to develop from a single cell in which mutations and/or epigenetic events have modified the function of genes responsible for cellular

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. 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, © 2017 The Author(s). Licensee InTech. 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.

growth regulation. These events establish the malignant phenotype, and subsequent molecular events may lead to the emergence of malignant subclones with enhanced growth and metastatic potential [1]. Cancer cells, replicating inappropriately, eventually interfere with normal tissue and organism functions, cause morbidity and may ultimately prove fatal in the absence of effective therapy. The rate of growth of tumours including, breast tumours, is determined by a balance between cell proliferation and cell death; if the rate of proliferation exceeds that of death, tumour growth will occur. Not surprisingly, many of the genes involved in neoplasia turn out to be concerned with control of cell death, as well as with control of cell proliferation [2].

The genes involved in neoplasia are usually classified as oncogenes or tumour suppressor genes, depending on whether the affected gene has gained or lost function in its mutated form. In keeping with this model, breast cancer is the result of imbalance in complex regulatory controls of cellular development and growth. Genetic abnormalities detected in breast carcinomas include mutation and amplification of oncogenes, mutation of tumour suppressor genes, and loss of heterozygosity at certain chromosomal loci. Sex hormones, growth factors, oncogenes, and tumour suppressor genes all regulate gene expression and thereby influence growth and function of breast epithelial cells. Influences favouring cell proliferation or inhibiting cell death may promote tumour progression [3].

Breast carcinoma is phenotypically complex: carcinoma in situ and invasive carcinoma may coexist, as mixed histological types of invasive carcinoma, and infiltrating ductal carcinomas often contain areas with different grades of disease. This morphological heterogeneity mirrors molecular heterogeneity, as well as morphologically similar tumours may differ in genetic and metabolic processes, and specific genetic abnormalities may influence clinical outcome [4, 5].

Prognosis and likely responses to therapy are clinically important in breast cancer and many variables have been evaluated. Classical morphological variables including histological type, tumour size, grade, lymph node status, and whether or not there is blood or lymphatic vascular invasion remain the strongest predictors of tumour behaviour. Attempts to evaluate breast tumour prognosis from individual or combined expression of variables such as oestrogen and progesterone receptor, cell proliferation index, S-phase fraction, DNA ploidy, growth factors and their receptors, oncogenes, tumour suppressor genes, proteases, components of the plasminogen system, and cell cycle regulators have yet to match the clinical utility of the classical morphological factors [6].

The chief forms of carcinoma of the breast are breast cancers that are classified into those that have not penetrated the limiting basement membrane (noninvasive) and those that have (invasive). The WHO current classification [7] is illustrated in **Figure 1**.

Breast cancer is the most commonly diagnosed cancer in women with an increased incidence of 14.1 million new cases in year 2012 and high mortality rate about 8.2 million deaths all over the world [8]. The incidence rate is expected to increase by year 2020 to reach about double the rate in 2012 [9]. Young women aged 20–59 years are expected to suffer from breast cancer with increased rate of death from cancer among their age group [10].

**Figure 1.** Classification of cancer breast.

### **2. Breast cancer risk factors**

The transformation of the normal epithelium into carcinoma is a multistep process. Genetic background and environmental and dietary factors have a role in breast cancer development. In the normal breast tissue, there is a balance between negative and positive growth factors, so to develop, breast cancer requires loss or gain in some functions [5]. The following factors are thought to be related to breast cancer development:

#### **2.1. Age**

growth regulation. These events establish the malignant phenotype, and subsequent molecular events may lead to the emergence of malignant subclones with enhanced growth and metastatic potential [1]. Cancer cells, replicating inappropriately, eventually interfere with normal tissue and organism functions, cause morbidity and may ultimately prove fatal in the absence of effective therapy. The rate of growth of tumours including, breast tumours, is determined by a balance between cell proliferation and cell death; if the rate of proliferation exceeds that of death, tumour growth will occur. Not surprisingly, many of the genes involved in neoplasia turn out to be concerned with control of cell death, as well as with

The genes involved in neoplasia are usually classified as oncogenes or tumour suppressor genes, depending on whether the affected gene has gained or lost function in its mutated form. In keeping with this model, breast cancer is the result of imbalance in complex regulatory controls of cellular development and growth. Genetic abnormalities detected in breast carcinomas include mutation and amplification of oncogenes, mutation of tumour suppressor genes, and loss of heterozygosity at certain chromosomal loci. Sex hormones, growth factors, oncogenes, and tumour suppressor genes all regulate gene expression and thereby influence growth and function of breast epithelial cells. Influences favouring cell proliferation or inhib-

Breast carcinoma is phenotypically complex: carcinoma in situ and invasive carcinoma may coexist, as mixed histological types of invasive carcinoma, and infiltrating ductal carcinomas often contain areas with different grades of disease. This morphological heterogeneity mirrors molecular heterogeneity, as well as morphologically similar tumours may differ in genetic and metabolic processes, and specific genetic abnormalities may influence clinical

Prognosis and likely responses to therapy are clinically important in breast cancer and many variables have been evaluated. Classical morphological variables including histological type, tumour size, grade, lymph node status, and whether or not there is blood or lymphatic vascular invasion remain the strongest predictors of tumour behaviour. Attempts to evaluate breast tumour prognosis from individual or combined expression of variables such as oestrogen and progesterone receptor, cell proliferation index, S-phase fraction, DNA ploidy, growth factors and their receptors, oncogenes, tumour suppressor genes, proteases, components of the plasminogen system, and cell cycle regulators have yet to match the clinical utility of the classical

The chief forms of carcinoma of the breast are breast cancers that are classified into those that have not penetrated the limiting basement membrane (noninvasive) and those that have

Breast cancer is the most commonly diagnosed cancer in women with an increased incidence of 14.1 million new cases in year 2012 and high mortality rate about 8.2 million deaths all over the world [8]. The incidence rate is expected to increase by year 2020 to reach about double the rate in 2012 [9].  The WHO current classification [7] is illustrated in **Figure 1**.

with increased rate of death from cancer among their age group [10].

control of cell proliferation [2].

344 Breast Cancer - From Biology to Medicine

outcome [4, 5].

morphological factors [6].

iting cell death may promote tumour progression [3].

The increased incidence of breast cancer with age may reflect the accumulation of somatic mutation. Early menarche and late menopause prolong the exposure to ovarian hormones and are associated with a higher incidence of breast cancer. There is some evidence that breast cancer in younger women is more aggressive than in older women, consistent with a more rapidly evolving disease declaring itself sooner clinically [11].

#### **2.2. Genetic factors**

Complex acquired genetic alterations are considered to cause breast cancer, and genetic abnormalities in the premalignant and malignant breast epithelium are likely to have a causal role [12]. That most breast cancers are due to acquired mutations is implied by the fact that only 5% of breast cancer patients have a strong family history indicating inheritance of tumour-promoting mutations in the germ line. Inherited early-onset breast cancer is largely attributable to two genes, BRCA1 and BRCA2. Li-Fraumeni syndrome, ataxia telangiectasia, and Cowden's disease are also associated with increased risk of breast cancer [13].

#### **2.3. Hormonal status**

Breast cancer risk appears to increase with exposure to mammotropic hormones, mainly oestrogen, progesterone, prolactin, and insulin-like growth factor 1 during adolescence and adult life. This may be explained by an increased epithelial cell population at risk during the preinitiation stage, affecting clonal expansion and modulating growth enhancement in subclinical tumours. Estrogen is a dominant influence on breast growth, but its role depends on oestrogen receptor (ER) expression in the target tissues. Recently, it has been suggested that overexpression of oestrogen receptors in the normal breast epithelium increases breast cancer risk in women [5].

#### **2.4.  In benign breast neoplasia, inactivation of tumour suppressor genes may occur and loss of heterozygosity is also reported. Ductal and lobular carcinomas in situ have a partly malignant morphological phenotype, lacking the ability to invade and metastasize, but are associated with elevated invasive cancer risk. Other lesions associated with abnormal cell proliferation are also associated with more modestly elevated cancer risk, notably the atypical hyperplasia (ductal and lobular) and florid hyperplasia of usual type (that is, without atypia). Frequent coexistence of premalignant lesions with invasive breast cancer is consistent with progression from these lesions to cancer, but there are many controversies in this area, and clonal relationships are not always clear [4].

### **3. Carcinogenesis and pathogenesis of breast cancer**

The prognosis of breast cancer is variable and affected by the heterogeneity of breast cancer, different pattern of breast subtypes, and aggressive genetic behaviour. All these factors may be associated with worsen patient outcome if accumulated with effect of hormonal status, and bilateral oophorectomy may improve prognosis in breast cancer.  Aggressive behaviour of breast cancer is due to collision of biologically active tumour and genetic abnormalities, but targeted interventions may improve the survival rate and patient outcome [3].

Estrogen has a crucial role in many tumours including ovaries, endometrium, and mammary gland cancers and also prostate cancer. Estrogen is linked to enhanced proliferation, decreased apoptosis, and DNA damage in breast cancer. Several experiments on animals have demonstrated that estradiol administration increased the risk of breast cancer, while antioestrogen agents had an opposite effect. Response of breast cancer to antioestrogen therapy after the confirmed presence of high percentage of hormonal receptors is defined as hormonaldependent breast cancer [15].

The presented framework of circulating tumour cell (CTC) biology and classification of CTC assays might help to structure this dynamic field of translational cancer research. Better insights into the biology of CTCs will further improve CTC assay development [1, 16].  Therefore, CTC belonging to breast cancer requires special detection approach. Specific profile for CTCs could be targeted for successful detection of complex aggressive breast cancer [17].

Tumorigeneses are postulated by many research works due to multiple steps and may start as chronic disease and processed to cancer. Intervention and prevention of these steps before cancer emerge is a good chance for reducing breast cancer risk. Adjuvant therapies as tamoxifen are effective and safe in significantly reducing and preventing molecular changes that lead to cancer. Those targeted hormonal therapies are very important to stop invasion and metastasis of tumour cells. Blocking DNA mutation is also initiated by using micronutrients and gene therapy to target abnormal pathways that claimed to had a role in carcinogenesis [5].

### **4. Gene expression profiling of breast cancer**

That most breast cancers are due to acquired mutations is implied by the fact that only 5% of breast cancer patients have a strong family history indicating inheritance of tumour-promoting mutations in the germ line. Inherited early-onset breast cancer is largely attributable to two genes, BRCA1 and BRCA2. Li-Fraumeni syndrome, ataxia telangiectasia, and Cowden's disease are also

Breast cancer risk appears to increase with exposure to mammotropic hormones, mainly oestrogen, progesterone, prolactin, and insulin-like growth factor 1 during adolescence and adult life. This may be explained by an increased epithelial cell population at risk during the preinitiation stage, affecting clonal expansion and modulating growth enhancement in subclinical tumours. Estrogen is a dominant influence on breast growth, but its role depends on oestrogen receptor (ER) expression in the target tissues. Recently, it has been suggested that overexpression of oestrogen receptors in the normal breast epithelium increases breast cancer risk in women [5].

Clear evidence exists that certain subtypes of benign breast disease are associated with breast cancer. In benign breast neoplasia, inactivation of tumour suppressor genes may occur and loss of heterozygosity is also reported. Ductal and lobular carcinomas in situ have a partly malignant morphological phenotype, lacking the ability to invade and metastasize, but are associated with elevated invasive cancer risk. Other lesions associated with abnormal cell proliferation are also associated with more modestly elevated cancer risk, notably the atypical hyperplasia (ductal and lobular) and florid hyperplasia of usual type (that is, without atypia). Frequent coexistence of premalignant lesions with invasive breast cancer is consistent with progression from these lesions to cancer, but there are many controversies in this area, and

The prognosis of breast cancer is variable and affected by the heterogeneity of breast cancer, different pattern of breast subtypes, and aggressive genetic behaviour. All these factors may be associated with worsen patient outcome if accumulated with effect of hormonal status, and bilateral oophorectomy may improve prognosis in breast cancer.  Aggressive behaviour of breast cancer is due to collision of biologically active tumour and genetic abnormalities, but targeted interventions may improve

Estrogen has a crucial role in many tumours including ovaries, endometrium, and mammary gland cancers and also prostate cancer. Estrogen is linked to enhanced proliferation,

associated with increased risk of breast cancer [13].

**2.3. Hormonal status**

346 Breast Cancer - From Biology to Medicine

**2.4. Previous benign breast disease**

clonal relationships are not always clear [4].

the survival rate and patient outcome [3].

**3. Carcinogenesis and pathogenesis of breast cancer**

Management of breast cancer depends on clinicopathologic parameters including age, stage, hormonal status, and Ki67 status. Alteration of molecular genetic character including alterations at DNA, RNA, and the protein functional changes contributes to oncogenesis. However, epigenetic changes and regulatory or transcriptional molecules as snRNA, siRNA, and miRNA may be other significantly contributing molecules as well. Successful therapy depends on hormone receptor and human epidermal growth factor receptor 2 (Her2) pathway by analysing their immunohistochemical expression. Failure to respond to the traditional treatment increases morbidity and mortality, and so further discovery of molecular variation in individuals could help in classifying breast cancer subtypes. Clonal analysis of different breast cancer types to understand molecular modifications and genetic expression help in producing full accurate histopathological diagnosis [18].

In the past decade, where breast cancer is clustered in families due to a common genetic factor BRCA1 or BRCA2 mutation, environmental factors shared between relatives may also be relevant. There has been progress in development of new therapeutic approaches that target these BRCA1 and BRCA2 cancer susceptibility genes, which led to loss of functional mutations in either BRCA1 or BRCA2 [19].

Recent delivery of nucleic acid mimics and therapeutic-based miRNA could be used for nanodelivery of target therapy to specific site. In addition, miRNA were recently studied as biological biomarkers in breast cancer, specifically for diagnosis, predicting cancer behavior and outcome [20].

#### **5. Hormone receptors**

The breast epithelium undergoes hyperplasia or involution in response to hormone supplementation or withdrawal, and oestrogen and progesterone receptors in the breast tissue mediate proliferative effects. Certain genetic alterations (alteration to the DNA-binding domain) are associated with high overall tumour grade and lack of steroid hormone receptors; an inverse correlation between both receptor expression and nuclear anaplasia also indicates a relation with cellular differentiation. Steroid hormone receptor expression in breast neoplasia has prognostic value. Moreover, there is an independent correlation between oestrogen and progesterone receptor status in breast cancer and tumour progression [21, 22].

#### **5.1. Oestrogen receptors (ER) and progesterone receptors (PR)**

Estrogen and progesterone receptors are unique signature to define personalized therapy of breast cancer, and their genetic expression may contribute to breast cancer management through using antioestrogen-targeted therapy. If mutated DNA is spilled from dying cancerous cells into the blood stream, it will become habitant into lymphatic or blood channels and socalled circulating tumor cells. Circulating tumour cells are capable of stimulating other tissues towards continuous proliferation and could be a tool to measure tumour power and ability to enhance metastases [18]. Further research is mandatory to identify those patients at high risk of breast cancer and to understand method of optimization of circulating tumour cell measurement. In addition, there is progressive need for clinical evolution of new agents and targeted therapy, especially to whom with BRCA positive and triple-negative breast cancer patients [23].

#### **5.2. HER2**

The HER2/neu oncogene is located on chromosome 17 at band q 21. It is related to the cerbB-1 (EGFR, HER1) gene which encodes the epidermal growth factor receptor. In addition to its function as a growth factor receptor, it is involved in the regulation of cellular differentiation, adhesion, and motility. When HER2/neu gene is amplified and as a result HER2/neu protein is substantially overexpressed, it is very likely that this plays a role in tumour development and progression [3]. HER2 amplification and overexpression may provide prognostic and therapeutic information in breast cancer and predict resistance to adjuvant therapy. Amplification of this gene is associated with rapid proliferation, shorter disease-free survival, and poorer prognosis in both node-negative and node-positive ductal breast carcinomas and a risk factor for the development of distant metastases. It has true independent prognostic significance but is associated with hormone-independent tumours. Progress of personalized medicine does well for patients to depend on Herceptin treatment. The ultimate goal is to understand cancer behaviour and improve patient survival rate and treatment outcome [6]. Some of the currently utilized cancer biomarkers for breast cancer and their clinical significance are illustrated in **Table 1**.

**Table 1.**

### **6. Molecular targeting therapy and personalized medicine in breast cancer**

Recent delivery of nucleic acid mimics and therapeutic-based miRNA could be used for nanodelivery of target therapy to specific site. In addition, miRNA were recently studied as biological biomarkers in breast cancer, specifically for diagnosis, predicting cancer behavior and outcome [20].

The breast epithelium undergoes hyperplasia or involution in response to hormone supplementation or withdrawal, and oestrogen and progesterone receptors in the breast tissue mediate proliferative effects. Certain genetic alterations (alteration to the DNA-binding domain) are associated with high overall tumour grade and lack of steroid hormone receptors; an inverse correlation between both receptor expression and nuclear anaplasia also indicates a relation with cellular differentiation. Steroid hormone receptor expression in breast neoplasia has prognostic value. Moreover, there is an independent correlation between oestrogen

Estrogen and progesterone receptors are unique signature to define personalized therapy of breast cancer, and their genetic expression may contribute to breast cancer management through using antioestrogen-targeted therapy. If mutated DNA is spilled from dying cancerous cells into the blood stream, it will become habitant into lymphatic or blood channels and socalled circulating tumor cells. Circulating tumour cells are capable of stimulating other tissues towards continuous proliferation and could be a tool to measure tumour power and ability to enhance metastases [18]. Further research is mandatory to identify those patients at high risk of breast cancer and to understand method of optimization of circulating tumour cell measurement. In addition, there is progressive need for clinical evolution of new agents and targeted therapy, especially to whom with BRCA positive and triple-negative breast cancer patients [23].

The HER2/neu oncogene is located on chromosome 17 at band q 21. It is related to the cerbB-1 (EGFR, HER1) gene which encodes the epidermal growth factor receptor. In addition to its function as a growth factor receptor, it is involved in the regulation of cellular differentiation, adhesion, and motility. When HER2/neu gene is amplified and as a result HER2/neu protein is substantially overexpressed, it is very likely that this plays a role in tumour development and progression [3]. HER2 amplification and overexpression may provide prognostic and therapeutic information in breast cancer and predict resistance to adjuvant therapy. Amplification of this gene is associated with rapid proliferation, shorter disease-free survival, and poorer prognosis in both node-negative and node-positive ductal breast carcinomas and a risk factor for the development of distant metastases. It has true independent prognostic significance but is associated with hormone-independent tumours. Progress of personalized medicine does well for patients to depend on Herceptin treatment. The ultimate goal is to understand cancer behaviour and improve patient survival rate and treatment outcome [6].  Oestrogen receptors (ER) and progesterone receptors (PR)**

**5. Hormone receptors**

348 Breast Cancer - From Biology to Medicine

**5.2. HER2**

Breast cancer is a heterogeneous disease that encompasses subtypes characterized by specific molecular biomarkers: oestrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2) positive, and triple-negative (TNBC) which are ER, progesterone receptor (PR), and HER2 negative breast cancers [5]. Perfect diagnosis and detection of molecular abnormalities help in improvement of personalized therapies to block these mutations by targeted therapy. The best example is using HER2 expression by immunohistochemistry or gene amplification to develop accurate therapy with trastuzumab [24]. Trastuzumab is targeted against domain IV of HER2 [25], while pertuzumab (Perjeta) is targeted monoclonal antibody against the ligand domain II of HER2. Lapatinib is dual targeting therapy for both HER2 and epidermal growth factor receptor (EGFR 1), specifically against its intracellular domain; it acts as tyrosine kinase inhibitor (TKI) as well [26]. Patients on combined lapatinib and trastuzumab have better outcome and survival; such therapy could be assessed with HER2 expression [27]. In fact, combining the HER2 targeting therapies as a neoadjuvant or for metastatic late stage, as trastuzumab and lapatinib or pertuzumab, will significantly improve patient's outcome in comparison with single anti-HER2 therapy [28–30]. Using combined and synergistically acting therapy on the same target (HER2) would achieve better response because of the concomitant action on same receptor against two different epitopes, in addition to the likely deaddiction effect of target that may involve stimulation of the immune system [31].

#### **6.1. mechanistic target of rapamycin (mTOR)/PI3K/Akt-pathway inhibitors**

Preclinical studies of the effects of AZD2014 in breast cancer are promising steps to confirm anti-proliferative role of mTOR signalling. Targeting mTOR pathway could suppress the development and progression of cancer, specifically gastrointestinal malignancies and breast cancer. The functions of mTOR pathway are mainly targeted for growth signaling, nutrient status, and metabolism with recent undiscovered impact in obesity development [23].

#### **6.2. Therapeutic cancer vaccine**

The monoclonal antibody drugs encourage T cells to detect and destroy cancer and increase the ability of the immune system to respond to tumours. Application of therapeutic cancer vaccine stimulates tumour antigen aiming at activation of tumour-specific T cells [32]. Therapeutic cancer vaccines require the selection of appropriate antigens that are not prone to central immunological tolerance induction in the thymus. Each type of cancer has its own particular immune-suppressive mechanisms guided by information of the immune memory. Selection of the best vaccine and its antigen delivery points should take place and be applied in treatment properly [33]. Breast cancer has the advantage of being based on huge antigen pool for an individual tumour, thus using tumor based-vaccines in breast cancer stimulate the activation of polyclonal immune responses. However, tolerance could occur to the immune system for expression of these antibodies. Co-stimulation and sensitization of the immune molecules will deliver all signals needed for activating T cells under the effect of antigen-specific immune response. Cytokines in breast cancer could be an ideal example of the immune costimulatory molecules [34].

### **7. "Omics" and promising biomarkers in breast cancer**

Recently, with the merging of "omic" technologies such as genomics, proteomics, metabolomics, transcriptomics, etc., a great advancement has been achieved in the field of cancer biology with better understanding of carcinogenesis, cancer progression, metastasis, and target therapy [35]. Microarray, mass spectrometry, and sequencing techniques provide evolutionary era for promising cancer biomarkers [36]. Transcriptional profiling has been reported as a valuable tool for classification and determination of prognosis in patients of breast cancer [37, 38]. Apart from diagnosis, prediction of response to therapy, and prediction of breast cancer patients' outcomes, biomarkers may estimate risk assessment of getting cancer [39]. Genetic alterations in breast cancer or methylation of promoters of cancer-specific or associated genes will definitely linked to altered expression of certain proteins and may be used as emerging cancer biomarkers [40].

#### **7.1. Genomic biomarkers: MammaPrint and Oncotype DX**

MammaPrint is one of the emerging genomic assays that have been reported as prognostic biomarker, MammaPrint assay analyses 70 genes' expression signatures, and it is used to stratify patients into good or poor risk groups for recurrence [41]. Another example of promising genomic markers is the 21-gene signature assessing test, Oncotype DX. It is a quantitative realtime qRT-PCR-based assay, and both assays may provide physicians with very effective prognostic information and consequently would help in selecting early-stage hormone responsive breast cancer patients who will have a likelihood of disease recurrence [38]. Signatures for both assays include genes as ER, HER2, PR-regulated transcripts and proliferation-linked genes that mainly have been utilized as a very effective tool for assessing the probability of recurrence as well as for classifying patients accordingly into high-, intermediate- or low-risk groups for recurrence. In addition, Oncotype DX assay may be used for assessing response to tamoxifen therapy [42].

#### **7.2. Proteomics**

**6.1. mechanistic target of rapamycin (mTOR)/PI3K/Akt-pathway inhibitors**

**7. "Omics" and promising biomarkers in breast cancer**

**7.1. Genomic biomarkers: MammaPrint and Oncotype DX**

**6.2. Therapeutic cancer vaccine**

350 Breast Cancer - From Biology to Medicine

costimulatory molecules [34].

cancer biomarkers [40].

Preclinical studies of the effects of AZD2014 in breast cancer are promising steps to confirm anti-proliferative role of mTOR signalling. Targeting mTOR pathway could suppress the development and progression of cancer, specifically gastrointestinal malignancies and breast cancer. The functions of mTOR pathway are mainly targeted for growth signaling, nutrient

The monoclonal antibody drugs encourage T cells to detect and destroy cancer and increase the ability of the immune system to respond to tumours. Application of therapeutic cancer vaccine stimulates tumour antigen aiming at activation of tumour-specific T cells [32]. Therapeutic cancer vaccines require the selection of appropriate antigens that are not prone to central immunological tolerance induction in the thymus. Each type of cancer has its own particular immune-suppressive mechanisms guided by information of the immune memory. Selection of the best vaccine and its antigen delivery points should take place and be applied in treatment properly [33]. Breast cancer has the advantage of being based on huge antigen pool for an individual tumour, thus using tumor based-vaccines in breast cancer stimulate the activation of polyclonal immune responses. However, tolerance could occur to the immune system for expression of these antibodies. Co-stimulation and sensitization of the immune molecules will deliver all signals needed for activating T cells under the effect of antigen-specific immune response. Cytokines in breast cancer could be an ideal example of the immune

Recently, with the merging of "omic" technologies such as genomics, proteomics, metabolomics, transcriptomics, etc., a great advancement has been achieved in the field of cancer biology with better understanding of carcinogenesis, cancer progression, metastasis, and target therapy [35]. Microarray, mass spectrometry, and sequencing techniques provide evolutionary era for promising cancer biomarkers [36]. Transcriptional profiling has been reported as a valuable tool for classification and determination of prognosis in patients of breast cancer [37, 38]. Apart from diagnosis, prediction of response to therapy, and prediction of breast cancer patients' outcomes, biomarkers may estimate risk assessment of getting cancer [39]. Genetic alterations in breast cancer or methylation of promoters of cancer-specific or associated genes will definitely linked to altered expression of certain proteins and may be used as emerging

MammaPrint is one of the emerging genomic assays that have been reported as prognostic biomarker, MammaPrint assay analyses 70 genes' expression signatures, and it is used to stratify patients into good or poor risk groups for recurrence [41]. Another example of promising

status, and metabolism with recent undiscovered impact in obesity development [23].

Proteomic approach has been investigated through mass spectrometry, two-dimensional gel electrophoresis, and other strategies and successfully has identified promising markers for early diagnosis of ovarian cancer [43, 44]. In spite of being invalidated, their results have paved the path for applying the proteomic approach, via mass spectrometry, for identification of other biomarkers in serum in breast cancer [45] and nipple aspirate as well [46]. Moreover, a panel of proteins has also been identified by high-throughput antibody arrays' technique, and their levels were significantly increasing in malignant breast tissue when compared to normal tissue. Such panel included p53, MAP kinase 7, and casein kinase Ie and annexin [47]. In fact, recent proteomics techniques such as nano-techniques are evolutionally emerging and promising to overcome few limitations of the conventional techniques for identification of potential biomarkers for early detection of cancer; thus, these techniques have to be applied on larger scale of cancer patients and, more importantly, with standardized protocols in order to validate the potentially valuable biomarkers [48].

#### **7.3. DNA methylation**

DNA methylation is an example of DNA modification that could be detected and linked with the unique identity of that gene; thus, DNA methylation patterns differ between normal and tumour tissues, and hence, targeting candidate genes could be used to identify and detect cancer cells in the blood or body fluid [49]. Identification of DNA methylation mapping and assays has been applied to nipple aspirate as well for detection of cancer cells at early stage of breast cancer [50]. DNA methylation assessment has been investigated as a prognostic marker for breast cancer in serum, and it was reported that methylation of adenomatous polyposis coli (APC) gene and Ras association domain family 1 isoform A was significantly linked and independently associated with poor outcome in breast cancer patients [51].

#### **7.4. Circulating tumour cells (CTCs)**

Circulating tumour cells (CTCs) are detached or disseminated cells from solid tumours or their metastasis into circulation. It has been firstly detected in the bone marrow, called disseminated tumour cell (DTC) patients with early-stage breast cancer [52]. Once CTCs dislodged from cancerous tissues into circulation, they retain the proliferating capacity and ability to settle in other tissues. CTCs have the capability to proliferate and eventually forming metastasis. Hence, CTC could be a predictor marker for invasion and metastases [18]. Recently, CTCs were considered a dynamic prognostic marker whether in early- or late-metastasizing breast cancer cases [53]. Evaluation of CTCs might contribute for efficient therapy monitoring; in addition, expression profiles of CTCs may predict the likely responses to treatment. As well, assessment of the molecular features of them may be a pivotal step for the optimization of therapy [54].

### **8. Conclusion and prospective**

Breast cancer with its heterogeneous nature and complex behaviour in great needs requires potential biomarkers to improve screening, diagnosis, classification, prognosis, and prediction to therapies. Understanding biology of breast tumour cell, host immune defences, and the tumour microenvironment may allow early detection and recurrence in breast cancer patients.

Breast cancer patients, clinician, pharmaceutical companies, and targeted therapy developer in great needs for flexible, simple, and inexpensive tests with sharp accurate comprehensive diagnosis. The identified molecular aberrations could be arrested and held up by the corresponding targeted compounds, which are best exemplified by detection of HER2neu expression in breast cancer by immunohistochemistry and gene amplification tests for accurate treatment with trastuzumab. Molecular fingerprint for breast cancer generated to help medical practitioner and healthcare providers to focus on patient's prognosis and adopted the preferred best therapeutic option. Designation of distinctive incompatible genetic markers enhances shrinkage of toxic side effect from overuse of therapies and exaggerated gains to patients.

### **Author details**

Hala Fawzy Mohamed Kamel1,2\*, Hiba Saeed Bagader Al-Amodi1 and Hanan Mohamed AbdElmoneim3,4

### **References**

[3] Daly, B. and Olopade, O.I., *A perfect storm: how tumor biology, genomics, and health care delivery patterns collide to create a racial survival disparity in breast cancer and proposed interventions for change*. CA Cancer J Clin, 2015. **65**(3): p. 221–238.

Hence, CTC could be a predictor marker for invasion and metastases [18]. Recently, CTCs were considered a dynamic prognostic marker whether in early- or late-metastasizing breast cancer cases [53]. Evaluation of CTCs might contribute for efficient therapy monitoring; in addition, expression profiles of CTCs may predict the likely responses to treatment. As well, assessment of the molecular features of them may be a pivotal step for the optimization of therapy [54].

Breast cancer with its heterogeneous nature and complex behaviour in great needs requires potential biomarkers to improve screening, diagnosis, classification, prognosis, and prediction to therapies. Understanding biology of breast tumour cell, host immune defences, and the tumour microenvironment may allow early detection and recurrence in breast cancer patients. Breast cancer patients, clinician, pharmaceutical companies, and targeted therapy developer in great needs for flexible, simple, and inexpensive tests with sharp accurate comprehensive diagnosis. The identified molecular aberrations could be arrested and held up by the corresponding targeted compounds, which are best exemplified by detection of HER2neu expression in breast cancer by immunohistochemistry and gene amplification tests for accurate treatment with trastuzumab. Molecular fingerprint for breast cancer generated to help medical practitioner and healthcare providers to focus on patient's prognosis and adopted the preferred best therapeutic option. Designation of distinctive incompatible genetic markers enhances shrink-

age of toxic side effect from overuse of therapies and exaggerated gains to patients.

\*Address all correspondence to<EMAIL_ADDRESS><EMAIL_ADDRESS>
4 Pathology Department, Faculty of Medicine, El-Minia University, El-Minia, Egypt

1 Biochemistry Department, Faculty of Medicine, Umm AL Qura University, Makhha, Saudi

2 Medical Biochemistry Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt 3 Pathology Department, Faculty of Medicine, Umm AL Qura University, Makhha, Saudi Arabia

[1] Allison, K.H., *Molecular pathology of breast cancer: what a pathologist needs to know*. Am J

and Hanan Mohamed

Hala Fawzy Mohamed Kamel1,2\*, Hiba Saeed Bagader Al-Amodi1

**8. Conclusion and prospective**

352 Breast Cancer - From Biology to Medicine

**Author details**

AbdElmoneim3,4

Arabia

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### **Cardiac Toxicity of HER2-Directed Therapy in Women with Breast Cancer: Epidemiology, Etiology, Risk Factors, and Management Cardiac Toxicity of HER2-Directed Therapy in Women with Breast Cancer: Epidemiology, Etiology, Risk Factors, and Management**

Shahid Ahmed, Arooba Talat, Nayyer Iqbal, Mohamed Emara, Duc Le, Amir Sami and Jim Xiang Mohamed Emara, Duc Le, Amir Sami and Jim Xiang

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Shahid Ahmed, Arooba Talat, Nayyer Iqbal,

http://dx.doi.org/10.5772/66437

#### **Abstract**

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356 Breast Cancer - From Biology to Medicine

The HER2-targeted therapy have profoundly changed the outcomes of women with HER2-positive breast cancers. Trastuzumab and pertuzumab, HER2-targeting monoclonal antibodies, lapatinib and Neratinib, small molecule inhibitors of HER2 and the epidermal growth factor receptor, and ado-trastuzumab emtansine, a HER2-positive directed antibody drug conjugate, are approved for the treatment of HER2-positive breast cancer.

Cardiac toxicity is a known adverse effects of trastuzumab, and other HER2-directed therapy. In most cases it manifests as mild and reversible left ventricle dysfunction. Nevertheless, symptomatic heart failure is not rare. The incidence and severity of cardiac dysfunction is greatest among women who received HER2-directed therapy in combination with anthracycline-based therapy. In addition, a borderline low normal left ventricle ejection fraction; prior treatment with antihypertensive medication; and older age are other risk factors for trastuzumab-related cardiac dysfunction. HER2 signaling plays an important role in modulating myocardial response to treatment-related injury. Management of trastuzumab and the other HER2 targeted treatment-related cardiac dysfunction has two key components: withdrawal of HER2-directed therapy and treatment of underlying cardiac dysfunction. A multidisciplinary approach is recommended for an optimal outcome. This chapter reviews cardiac toxicity of trastuzumab and other HER2-directed therapy including epidemiology and pathophysiology of cardiac dysfunction, cardiac monitoring, treatment and prevention.

**Keywords:** breast cancer, HER2-directed therapy, cardiac toxicity, trastuzumab, pertuzumab, lapatinib, T-DM1, Neratinib

and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. 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, © 2017 The Author(s). Licensee InTech. 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.

### **1. Introduction**

Breast cancer is one of the most common cancers in women worldwide [1]. In 2012, nearly 1.7 million women were diagnosed with breast cancer. This represents about 12% of all new cancer cases and 25% of all cancers in women [2]. Approximately 20–25% of all breast cancers overexpressed the human epidermal growth factor receptor-2 (HER2). This protein is a member of the HER family of transmembrane receptor tyrosine kinases and is located at the cell surface. HER2 is involved in cellular growth and differentiation, and its overexpression has been associated with adverse prognosis. Prior to the development of HER2-targeted therapy, women with HER2-positive breast cancer had poor outcomes. However, access to HER2-directed therapy including monoclonal antibodies, small molecule inhibitors, and antibody-drug conjugates in the management of early and advanced breast cancer has transformed the natural history of HER2-positive breast cancer [3, 4]. HER2-targeted therapy alone or in combination with chemotherapy has been associated with improvements in response rate, disease control rates, and overall survival in HER2-positive metastatic breast cancer [3–5]. Combination of HER2-targeted agents including dual HER2 blockade and selected delivery of potent chemotherapeutic agent along with HER2 inhibition are new therapeutic approaches that in many women have transformed metastatic HER2-positive breast cancer into a chronic disease. More importantly, HER2 blockade in early-stage breast cancer has resulted in lower recurrence and mortality [3, 6].

As the outcomes of women with HER2-positive breast cancer have improved, increasingly attention has been directed toward minimizing both acute and chronic treatment-related toxicities. Cardiac toxicity is a known adverse effect of trastuzumab and other HER2 directed therapy [7, 8]. In most cases, it manifests as mild and reversible left ventricle dysfunction. Nevertheless, overt heart failure is not unusual. Serial monitoring of cardiac function is recommended for women treated with HER2-directed therapy. In women with treatment-related cardiac dysfunction, trastuzumab and other HER2-directed therapy interruptions and treatment of cardiac dysfunction are recommended. This chapter provides a summary of efficacy of HER2-directed therapy in breast cancer and reviews the incidence, pathophysiology, risk factors, monitoring, and management and prevention of HER2-targeted treatment-related cardiac dysfunction.

### **2. Efficacy of HER2‐directed therapy**

The current HER2-directed treatments for women with HER2-positive breast cancer include monoclonal antibodies, small molecule inhibitors, and antibody-drug conjugates (**Table 1**). Trastuzumab is the prototype humanized monoclonal antibody directed against the extracellular domain of human epidermal growth factor receptor-2 [9]. It was first evaluated in women with HER2-positive metastatic breast cancer. The combination of trastuzumab and chemotherapy resulted in improvement in progression-free and overall survival compared with chemotherapy alone, in women with HER2-positive metastatic breast cancer [5]. A Cochrane review assessed efficacy and safety of trastuzumab in seven trials, involving 1497 women with advanced breast cancer [10]. The combined hazard ratios (HRs) for overall survival and progression-free survival favored the trastuzumab-containing regimens (HR 0.82, 95% confidence interval (CI) 0.71–0.94, *p*-value = 0.004; and HR 0.61, 95% CI 0.54–0.70, *p*-value < 0.00001, respectively).

**1. Introduction**

358 Breast Cancer - From Biology to Medicine

resulted in lower recurrence and mortality [3, 6].

HER2-targeted treatment-related cardiac dysfunction.

**2. Efficacy of HER2‐directed therapy**

Breast cancer is one of the most common cancers in women worldwide [1]. In 2012, nearly 1.7 million women were diagnosed with breast cancer. This represents about 12% of all new cancer cases and 25% of all cancers in women [2]. Approximately 20–25% of all breast cancers overexpressed the human epidermal growth factor receptor-2 (HER2). This protein is a member of the HER family of transmembrane receptor tyrosine kinases and is located at the cell surface. HER2 is involved in cellular growth and differentiation, and its overexpression has been associated with adverse prognosis. Prior to the development of HER2-targeted therapy, women with HER2-positive breast cancer had poor outcomes. However, access to HER2-directed therapy including monoclonal antibodies, small molecule inhibitors, and antibody-drug conjugates in the management of early and advanced breast cancer has transformed the natural history of HER2-positive breast cancer [3, 4]. HER2-targeted therapy alone or in combination with chemotherapy has been associated with improvements in response rate, disease control rates, and overall survival in HER2-positive metastatic breast cancer [3–5]. Combination of HER2-targeted agents including dual HER2 blockade and selected delivery of potent chemotherapeutic agent along with HER2 inhibition are new therapeutic approaches that in many women have transformed metastatic HER2-positive breast cancer into a chronic disease. More importantly, HER2 blockade in early-stage breast cancer has

As the outcomes of women with HER2-positive breast cancer have improved, increasingly attention has been directed toward minimizing both acute and chronic treatment-related toxicities. Cardiac toxicity is a known adverse effect of trastuzumab and other HER2 directed therapy [7, 8]. In most cases, it manifests as mild and reversible left ventricle dysfunction. Nevertheless, overt heart failure is not unusual. Serial monitoring of cardiac function is recommended for women treated with HER2-directed therapy. In women with treatment-related cardiac dysfunction, trastuzumab and other HER2-directed therapy interruptions and treatment of cardiac dysfunction are recommended. This chapter provides a summary of efficacy of HER2-directed therapy in breast cancer and reviews the incidence, pathophysiology, risk factors, monitoring, and management and prevention of

The current HER2-directed treatments for women with HER2-positive breast cancer include monoclonal antibodies, small molecule inhibitors, and antibody-drug conjugates (**Table 1**). Trastuzumab is the prototype humanized monoclonal antibody directed against the extracellular domain of human epidermal growth factor receptor-2 [9]. It was first evaluated in women with HER2-positive metastatic breast cancer. The combination of trastuzumab and chemotherapy resulted in improvement in progression-free and overall survival compared with chemotherapy alone, in women with HER2-positive metastatic breast cancer [5]. A Cochrane review assessed efficacy and safety of trastuzumab in seven trials, involving 1497 women

**Table 1.** List of current HER2-directed targeted drugs that are approved for the management of HER2-positive breast cancer.

Later trastuzumab was evaluated in women with early-stage breast cancer, in both adjuvant and neoadjuvant settings, and demonstrated improvement in disease-free and overall survival. A Cochrane review evaluated efficacy and toxicity of trastuzumab in eight studies involving 11,991 women with early-stage breast cancer [11]. The combined HRs for overall survival and disease-free survival significantly favored the trastuzumab-containing regimens (HR 0.66; 95% CI 0.57–0.77, *p*-value < 0.00001; and HR 0.60; 95% CI 0.50–0.71, *p*-value < 0.00001, respectively).

Lapatinib is a dual EGFR/HER2 reversible tyrosine kinase inhibitor that suppresses the downstream signaling involving MAPK/Erk1/2 and PI3K/Akt pathways by blocking both HER1 and HER2 [15]. Lapatinib has demonstrated efficacy in HER2-positive advanced breast cancer [16]. In addition, it has been assessed in both adjuvant and neoadjuvant settings in women with early-stage breast cancer. However, overall the data suggest that lapatinib in early-stage breast cancer is inferior compared with trastuzumab [3, 17].

Pertuzumab is a humanized monoclonal antibody that binds to the extracellular domain II of HER2. It inhibits ligand-dependent HER2-HER3 dimerization and reduces signaling via intracellular pathways such as PI3K/Akt [18]. Pertuzumab has limited antitumor clinical activity alone, but it is a very good synergistic drug when combined with trastuzumab and has demonstrated benefit in combination with trastuzumab in the treatment of both early (neoadjuvant setting) and advanced HER2-positive breast cancer [3, 19, 20]. In the neoadjuvant setting, the pooled pathological complete response rate in the dual anti-HER2 therapy group was 54.8% compared with 36% in the monotherapy group when used in combination with chemotherapy (relative risk [RR], 1.56; 95% CI 1.23–1.97; *p*-value < 0.001). In the metastatic setting, dual anti-HER2 therapy demonstrated significant benefits in both progression-free survival (HR, 0.71; 95% CI 0.62–0.81; *p*-value < 0.001) and overall survival (HR, 0.68; 95% CI 0.57–0.82; *p*-value < 0.001) [21].

Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of an antimicrotubule cytotoxic agent DM1 linked to trastuzumab [22]. In women with HER2-positive advanced breast cancer, who were previously treated with trastuzumab and a taxane, it has shown significant improvement in progression-free and overall survival compared with lapatinib plus capecitabine [23].

Neratinib is an irreversible binder of HER1, HER2, and HER3 receptors [22, 24] It has demonstrated efficacy in HER2-positive breast cancer that progress on trastuzumab [3]. In addition, 1 year of neratinib following adjuvant chemotherapy and trastuzumab in women with HER2-positive breast cancer has been associated with modest improvement in disease-free survival [25].

In summary, over the past 15 years, HER2-directed therapy has revolutionized the management of HER2-positive breast cancer. In women with early-stage cancer, neoadjuvant and adjuvant HER2-directed therapies have substantially improved the disease-free and overall survival. Likewise, for many women, HER2-targeted therapy has transformed HER2-positive advanced breast cancer into a chronic disease. For example, median overall survival of women with HER2-positive advanced cancer has improved from 20.3 months reported by Slamon et al. in the first randomized trial using trastuzumab with chemotherapy to 48 months with the use of triple combination of pertuzumab, trastuzumab, and docetaxel [5, 26].

### **3. Cardiac safety of HER2‐directed therapy**

#### **3.1. Trastuzumab**

Trastuzumab-related cardiac dysfunction incidence varies according to the underlying treated population and the definition of cardiac toxicity used in the clinical trials. In the pivotal clinical trial that evaluated trastuzumab in combination with chemotherapy (anthracycline or taxane) in women with HER2-positive metastatic breast cancer, a high rate of cardiac dysfunction was noted, especially when trastuzumab was given in combination with an anthracycline-based chemotherapy [5]. In this trial, cardiac dysfunction was observed in 27% of the women who received an anthracycline, cyclophosphamide, and trastuzumab; 8% of the women who received an anthracycline and cyclophosphamide alone; 13% of the women who received paclitaxel and trastuzumab; and only 1% of the women who received paclitaxel alone. Among these women, the incidence of cardiac dysfunction of New York Heart Association class III or IV was 16% among women who were treated with an anthracycline, cyclophosphamide, and trastuzumab; 3% among women who received an anthracycline and cyclophosphamide alone; 2% among women who received paclitaxel and trastuzumab; and 1% among those who were treated with paclitaxel alone (**Table 2**). Given a high risk of symptomatic heart failure with the concomitant use of trastuzumab with anthracycline, in all adjuvant breast cancer trials, trastuzumab was only used after anthracyclines or with anthracycline-free chemotherapy. A Cochrane review assessed efficacy and safety of trastuzumab in seven trials, involving 1497 women with advanced breast cancer [10]. Trastuzumab increased the risk of congestive heart failure (CHF) (RR 3.49, 90% CI 1.88–6.47, *p*-value = 0.0009) and left ventricular ejection fraction (LVEF) decline (RR 2.65, 90% CI 1.48–4.74, *p*-value = 0.006).

Pertuzumab is a humanized monoclonal antibody that binds to the extracellular domain II of HER2. It inhibits ligand-dependent HER2-HER3 dimerization and reduces signaling via intracellular pathways such as PI3K/Akt [18]. Pertuzumab has limited antitumor clinical activity alone, but it is a very good synergistic drug when combined with trastuzumab and has demonstrated benefit in combination with trastuzumab in the treatment of both early (neoadjuvant setting) and advanced HER2-positive breast cancer [3, 19, 20]. In the neoadjuvant setting, the pooled pathological complete response rate in the dual anti-HER2 therapy group was 54.8% compared with 36% in the monotherapy group when used in combination with chemotherapy (relative risk [RR], 1.56; 95% CI 1.23–1.97; *p*-value < 0.001). In the metastatic setting, dual anti-HER2 therapy demonstrated significant benefits in both progression-free survival (HR, 0.71; 95% CI 0.62–0.81; *p*-value < 0.001) and overall survival (HR, 0.68; 95% CI 0.57–0.82; *p*-value < 0.001) [21]. Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of an antimicrotubule cytotoxic agent DM1 linked to trastuzumab [22]. In women with HER2-positive advanced breast cancer, who were previously treated with trastuzumab and a taxane, it has shown significant improvement in progression-free and overall survival compared with lapa-

Neratinib is an irreversible binder of HER1, HER2, and HER3 receptors [22, 24] It has demonstrated efficacy in HER2-positive breast cancer that progress on trastuzumab [3]. In addition, 1 year of neratinib following adjuvant chemotherapy and trastuzumab in women with HER2-positive breast cancer has been associated with modest improvement in disease-free

In summary, over the past 15 years, HER2-directed therapy has revolutionized the management of HER2-positive breast cancer. In women with early-stage cancer, neoadjuvant and adjuvant HER2-directed therapies have substantially improved the disease-free and overall survival. Likewise, for many women, HER2-targeted therapy has transformed HER2-positive advanced breast cancer into a chronic disease. For example, median overall survival of women with HER2-positive advanced cancer has improved from 20.3 months reported by Slamon et al. in the first randomized trial using trastuzumab with chemotherapy to 48 months with the use of triple combination of pertuzumab, trastuzumab, and

Trastuzumab-related cardiac dysfunction incidence varies according to the underlying treated population and the definition of cardiac toxicity used in the clinical trials. In the pivotal clinical trial that evaluated trastuzumab in combination with chemotherapy (anthracycline or taxane) in women with HER2-positive metastatic breast cancer, a high rate of cardiac dysfunction was noted, especially when trastuzumab was given in combination with an anthracycline-based chemotherapy [5]. In this trial, cardiac dysfunction was observed in

tinib plus capecitabine [23].

360 Breast Cancer - From Biology to Medicine

survival [25].

docetaxel [5, 26].

**3.1. Trastuzumab**

**3. Cardiac safety of HER2‐directed therapy**

**Table 2.** New York association and Canadian Cardiovascular Society functional classifications.

In the major adjuvant trastuzumab clinical trials, the rates of symptomatic CHF varied from 0.6 to 4.1%, whereas the rates of symptomatic or minimally symptomatic reduction in LVEF ranged from 4 to 34% (**Table 3**). The Herceptin Adjuvant (HERA) trial compared 1 or 2 years of trastuzumab given once every 3 weeks with observation in women with HER2-positive breast cancer. The incidence of trastuzumab discontinuation due to cardiac disorders was 4.3% [8]. The incidence of cardiac end points was higher in the trastuzumab group compared with observation: severe CHF, 0.60% compared with 0.06%; symptomatic CHF, 2.15% compared with 0.12%; and confirmed significant LVEF drops, 3.04% compared with 0.53%. Most women with cardiac dysfunction recovered in fewer than 6 months.

The National Surgical Adjuvant Breast and Bowel Project trial B-31 compared doxorubicin and cyclophosphamide (AC) followed by paclitaxel with AC followed by paclitaxel plus 52 weeks of trastuzumab beginning concurrently with paclitaxel in women with nodepositive, HER2-positive breast cancer [27]. Among women with normal post-AC LVEF

A: anthracycline; C: cyclophosphamide; T: taxane; H: trastuzumab; Ca: carboplatin; V: vinorelbine; F: 5-flourouracil; E: epirubicin; n/a: information not available; HF: heart failure; LV0EF: left ventricular heart failure; MUGA: multi-gated acquisition scan.

No prior anthracycline before H exposure; H exposure limited to 9 weeks.

b Measured from baseline.

a

c 6.7% did not receive H after A due to unacceptable drops in LVEF.

dIncluded a nonanthracycline arm.

e96% of chemotherapy was A containing. **Table 3.** Rates of asymptomatic and symptomatic cardiac dysfunction in various adjuvant trastuzumab phase 3 clinical trials. who began post-AC treatment, 5 of 814 (0.006%) women in the control group developed a cardiac event compared with 31 of 850 (0.036%) women treated with trastuzumab. The difference in cumulative incidence at 3 years was 3.3% (4.1% for trastuzumab-treated women minus 0.8% for control patients; 95% CI 1.7–4.9%). Twenty-seven of the 31 patients in the trastuzumab arm have been followed for ≥6 months after diagnosis of a CE; 26 were asymptomatic at last assessment; and 18 remained on cardiac medication. Fourteen percent of patients discontinued trastuzumab because of asymptomatic decreases in LVEF; 4% discontinued trastuzumab because of symptomatic cardiotoxicity.

In the North Central Cancer Treatment Group N9831 adjuvant breast cancer trial, women with HER2-positive operable breast cancer were randomly assigned to AC followed by either weekly paclitaxel (arm A); paclitaxel then trastuzumab (arm B); or paclitaxel plus trastuzumab then trastuzumab alone (arm C) [28]. There were 1944 women with satisfactory or no LVEF evaluation who proceeded to post-AC therapy. Cardiac events (CHF or cardiac death) were as followed: arm A, n = 3; arm B, n = 19; and arm C, n = 19 with 3-year cumulative incidences of 0.3, 2.8, and 3.3%, respectively. Incidence of asymptomatic LVEF decreases requiring holding trastuzumab was 8–10%; LVEF recovered and trastuzumab were restarted in approximately 50%.

The Breast Cancer International Research Group randomly assigned 3222 women with HER2-positive early-stage breast cancer to receive doxorubicin and cyclophosphamide followed by docetaxel every 3 weeks (AC-T), the same regimen plus 52 weeks of trastuzumab (AC-T plus trastuzumab), or docetaxel and carboplatin plus 52 weeks of trastuzumab (TCH) [6]. The incidence of congestive heart failure in the two trastuzumab-containing regimens was higher in the group receiving AC-T plus trastuzumab (2.0%) than in the AC-T group (0.7%) or the TCH group (0.4%); the incidence with AC-T plus trastuzumab as compared with TCH was increased by a factor of five. In addition, a significant difference in sustained, subclinical loss of mean LVEF (defined as >10% relative loss), was observed in the group receiving AC-T plus trastuzumab, as compared with the TCH group (18.6 versus 9.4%, *p*-value < 0.001), with a rate of 11.2% in the AC-T group. Of 194 of the 1042 patients (19%) who had a relative reduction in LVEF of more than 10% in the group receiving AC-T plus trastuzumab, the decrease persisted for at least 4 years after randomization in 33% of the women.

The FinHer investigators randomly assigned 1010 women to receive three cycles of docetaxel or vinorelbine, followed by three cycles of fluorouracil, epirubicin, and cyclophosphamide (FEC). The 232 women with HER2-positive cancer were further assigned to receive or not to receive nine weekly trastuzumab infusions [12]. The incidence of symptomatic heart failure among the HER2-positive women was 0.9% (one patient) with trastuzumab and 1.7% (two patients) without trastuzumab. The incidence of absolute declines in LVEF >20% points from baseline was 6.8% with trastuzumab and 10.5% without trastuzumab [12, 29].

The Cochrane review evaluated toxicity of trastuzumab in eight studies involving 11,991 women with early-stage breast cancer [11]. Trastuzumab significantly increased the risk of CHF (RR 5.11; 90% CI 3.00–8.72, *p*-value < 0.00001) and LVEF (RR 1.83; 90% CI 1.36–2.47, *p*-value = 0.0008).

**Trial**

FinHer12, 29

NSABP

2030

AC + TH + H

Grade III/IV HF or cardiac

death; or LVEF decrease >15

pointsb

versus AC + T

B-3127, 14

BCIRG 0066

3222

AC + T versus

Grade III/IV HF; cardiac

death; grade 3–4

arrhythmias; grade 3–4

ischemia/infarction; or LVEF

decrease >10 pointsb

Grade III/IV HF or cardiac

MUGA or echo at entry, after

5.8–10.4 versus

3.3 versus 2.8%

n/ab

versus 0.3%

4.0–7.8% versus

4.0–5.1%

7.1 versus 2.2%

0.6 versus 0.06%

4.3%

AC, and 6, 9, 18, and 21 months

after entry

death; or LVEF decrease >15

NCCTG

3505

AC + TH + H

versus AC + T + H

versus AC + T

pointsb

N983128, 14

HERA8, 13

5090

Adj chemoce ≥H

Severe HF; symptomatic HF;

LVEF (echo or MUGA) at

baseline, 3, 6, 12, 18, 24, 30, 36,

and 60 months

A: anthracycline; C: cyclophosphamide; T: taxane; H: trastuzumab; Ca: carboplatin; V: vinorelbine; F: 5-flourouracil; E: epirubicin; n/a: information not available; HF: heart

failure; LV0EF: left ventricular heart failure; MUGA: multi-gated acquisition scan.

No prior anthracycline before H exposure; H exposure limited to 9 weeks.

a b Measured from baseline.

c d e **Table 3.**

Rates of asymptomatic and symptomatic cardiac dysfunction in various adjuvant trastuzumab phase 3 clinical trials.

Included a nonanthracycline arm.

96% of chemotherapy was A containing.

6.7% did not receive H after A due to unacceptable drops in LVEF.

or LVEF decrease >10 points

versus Adj chemo

alone

AC + TH + H

versus TCaHd

232

V or T + H versus

Myocardial infarction; HF;

Echo or MUGA before

chemotherapy, after FEC,

and 12 and 36 months after

chemotherapy

MUGA 3 weeks, 6, and

34 versus 17%

4.1 versus 0.8%

19%c

9 months after end of initial

AC, and 3 months after last

trastuzumab dose

After AC, after second

11 versus 19%

0.7 versus 2.0%

n/a

versus 0.4%

versus 9%

dose of docetaxel, at end of

chemotherapy, and 3, 12, and

36 months after randomization

or LVEF decrease >15 points

V or Ta ≥FEC × 3

**N**

**Design**

**Definition of severe**