5. Genomic markers, prognosis, and personalized treatment

In the past, breast cancers were simply treated based on some clinicopathological features, such as tumor size, lymph node status, patients age and menopausal status, and tumor biomarkers such as ER, PR, and HER2/neu. Then, systemic chemotherapy was applied nearly universally to locally advanced breast cancers regardless of their biomolecular profile, and to about 60% of early breast cancers, but often without any significant effect on women prognosis [105]. As a consequence, a great debate has emerged about quality-of-life issues, acute and long-term side effects of systemic therapies, and the cost of unnecessary treatments [54, 106]. Therefore, in the last decades, quantitative approaches for prognosis prediction and treatment individualization have been developed, and genomic and molecular technologies are routinely applied to prevent overtreatments.

Recently, thanks to the increased level of knowledge regarding the molecular pathways and underlying genetic changes in breast cancer, the molecular signatures of gene expression have been correlated with breast cancer recurrence risk [7, 107, 108]. Anyway, their current clinical application is still limited due to reproducibility questions and the need for fresh or frozen tissue.

In this section, we discuss about susceptibility genes, the carriers of which results to have an increased breast cancer risk and consequently deserve a more frequent and specific screening, and about some signatures, which are usually used to predict breast cancer responsiveness to adjuvant and neoadjuvant therapies.

Mutations in BRCA1 and BRCA2 genes are the most frequent hereditary genetic aberrations in breast cancer and account for approximately half of all hereditary breast cancers. Initial estimates found BRCA1 mutations to be responsible for 45–90% of breast cancer cases in families with apparent autosomal dominant transmission of breast cancer, and this percentage rises if the median age at onset of breast cancer is younger than 45 years [120, 121]. Estimates of BRCA1 and BRCA2 mutation prevalence in unselected patients with breast cancer are in the

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Among BRCA1 mutation carriers, the estimated breast cancer risk is about 65%, the estimated risk of contralateral breast cancer occurrence results 60%, the cumulative risk of ovarian cancer varies between 27 and 45%, and there is also a significantly increased risk of fallopian tube, uterine and cervical cancer, as well as of male breast cancer, stomach, pancreatic, colon and

Among BRCA2 mutation carriers, the estimated lifetime breast cancer risk ranges between 45 and 84%, and that of ovarian cancer between 10 and 20% [123, 127]. There is an increased male breast cancer risk of about 6%, as well as an increased risk of prostate, pancreatic, stomach,

More than 500 coding region sequence variations have been detected in BRCA1 and 250 in BRCA2. Most unequivocally confirmed mutations reported to date are truncating mutations, adding little in the way of clues for defining functional regions. Although few mutations have been identified in either gene in sporadic breast cancers, a phenotype termed "BRCAness" exist, in which the BRCA1 and BRCA2 proteins act may be somehow disrupted also in sporadic cancer [129]. Finally, there are also some syndromes characterized by an increased

Although germline mutations in BRCA1 and BRCA2 confer a high risk of breast cancer, a great deal of variability has been observed in cancer risk among individuals, both between and within families, as many environmental or genetic factors can modify the penetrance of BRCA1 and BRCA2 mutations. The Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA), analyzing DNA and clinical data from approximately 10,000 BRCA1 and 5000 BRCA2 mutation carriers, demonstrated the role of a number of gene variants in affecting

The most important prognostic modifiers of BRCA1 and BRCA2 mutation carriers are prophylactic oophorectomy and the use of tamoxifen for chemoprevention, which approximately halve the breast cancer risk and decrease the risk of ovarian cancer by about 95% [131, 132].

In comparison with sporadic breast cancers, those related with high-penetrance susceptibility genes correlate with younger age at diagnosis, more aggressive tumor biological behavior, bilaterality, and the eventual coexistence of other cancers in the same individual or in other individuals of the same family, including ovarian, colon, prostate, pancreatic, and endometrial

Breast cancers arising in BRCA1 mutation carriers are frequently, although not exclusively, basal-like and ER-negative, probably because BRCA1 plays a crucial role in the transcriptional

gallbladder and bile duct cancers, and malignant melanoma [122, 128].

breast cancer susceptibility, which are discussed in the following section.

range of 2–3% [122].

testicular cancer [123–126].

the penetrance in mutation carriers [130].

cancers, as well as sarcomas and male breast cancer [133].

#### 5.1. BRCA1 and BRCA2

Inherited susceptibility to breast cancer has been hypothesized due to the discovery and characterization of a number of high-risk, relatively uncommon genes responsible for the clustering of breast cancer in certain families, which thereafter had a significantly increased risk in comparison with the general population [109, 110]. Many studies suggest that breast cancer susceptibility is transmitted in an autosomal dominant mendelian way [111], but the actual risk of developing breast cancer in a mutation carrier is based on the penetrance of the gene, which consists in the likelihood that the effect (phenotype) of a mutation (genotype) will become clinically apparent.

The BRCA1 gene was firstly identified in 1994 [112] and is localized on the 17th chromosome, whereas the BRCA2 gene was found some years after and is localized on the 13th chromosome [113]. The big size of these genes is important in the context of genetic testing because of the increased probability of mutations and the consequent technically demanding and costly mutations testing, but fortunately the use of modern next generation DNA sequencing is already overcoming these technical and cost issues. Moreover, the BRCA1 gene contains a large number of repetitive elements that facilitate the generation of large deletions and duplications.

BRCA1 is a nuclear protein with two important regions of sequence similarity with known functional motifs: a 42–amino-acid RING (Really Interesting New Gene) domain at the beginning of BRCA1 which binds zinc and is essential in cell growth and differentiation, and the BRCT (breast cancer-1 terminus) motif at the carboxyl terminus, which acts as a phosphoprotein docking motif and a transcriptional activation domain [114, 115]. BRCA2 is also a nuclear protein composed of the following major structural motifs: the eight tandem BRC repeats in the central portion of the protein, which mediates the critical interaction of BRCA2 and RAD51, the TR2 at the carboxyl terminus, which binds RAD51 exists, a single-strand and double-strand DNA binding domain in the C-terminus [113, 116].

Both BRCA1 and BRCA2 genes encode large proteins with multiple functions, which act mainly as tumor suppressor gene products, affecting transcription, cell cycle regulation, genome stability maintenance, and repair of doublestranded DNA breaks for protection of the genome during replication [117, 118]. In particular, BRCA1 prevents replication of damaged DNA by altering chromatin structure and nucleosome organization at the local site of damage, facilitates access by repair complexes, and promotes the use of the error-free repair pathway of homologous recombination-mediated repair rather than the error-prone process of nonhomologous end joining [118]. BRCA2 affects the choice between the two homologous recombination pathways in favor of the error-free one, by interacting with RAD51 [118]. When the wild-type BRCA1 or BRCA2 allele is lost, mutated, or silenced, a high degree of chromosome instability is observed and defective DNA repair may occur, with the consequent accumulation of additional mutations during replication and promotion of carcinogenesis [119].

Mutations in BRCA1 and BRCA2 genes are the most frequent hereditary genetic aberrations in breast cancer and account for approximately half of all hereditary breast cancers. Initial estimates found BRCA1 mutations to be responsible for 45–90% of breast cancer cases in families with apparent autosomal dominant transmission of breast cancer, and this percentage rises if the median age at onset of breast cancer is younger than 45 years [120, 121]. Estimates of BRCA1 and BRCA2 mutation prevalence in unselected patients with breast cancer are in the range of 2–3% [122].

and about some signatures, which are usually used to predict breast cancer responsiveness to

Inherited susceptibility to breast cancer has been hypothesized due to the discovery and characterization of a number of high-risk, relatively uncommon genes responsible for the clustering of breast cancer in certain families, which thereafter had a significantly increased risk in comparison with the general population [109, 110]. Many studies suggest that breast cancer susceptibility is transmitted in an autosomal dominant mendelian way [111], but the actual risk of developing breast cancer in a mutation carrier is based on the penetrance of the gene, which consists in the likelihood that the effect (phenotype) of a mutation (genotype) will

The BRCA1 gene was firstly identified in 1994 [112] and is localized on the 17th chromosome, whereas the BRCA2 gene was found some years after and is localized on the 13th chromosome [113]. The big size of these genes is important in the context of genetic testing because of the increased probability of mutations and the consequent technically demanding and costly mutations testing, but fortunately the use of modern next generation DNA sequencing is already overcoming these technical and cost issues. Moreover, the BRCA1 gene contains a large number of repetitive elements that facilitate the generation of large deletions and dupli-

BRCA1 is a nuclear protein with two important regions of sequence similarity with known functional motifs: a 42–amino-acid RING (Really Interesting New Gene) domain at the beginning of BRCA1 which binds zinc and is essential in cell growth and differentiation, and the BRCT (breast cancer-1 terminus) motif at the carboxyl terminus, which acts as a phosphoprotein docking motif and a transcriptional activation domain [114, 115]. BRCA2 is also a nuclear protein composed of the following major structural motifs: the eight tandem BRC repeats in the central portion of the protein, which mediates the critical interaction of BRCA2 and RAD51, the TR2 at the carboxyl terminus, which binds RAD51 exists, a single-strand and

Both BRCA1 and BRCA2 genes encode large proteins with multiple functions, which act mainly as tumor suppressor gene products, affecting transcription, cell cycle regulation, genome stability maintenance, and repair of doublestranded DNA breaks for protection of the genome during replication [117, 118]. In particular, BRCA1 prevents replication of damaged DNA by altering chromatin structure and nucleosome organization at the local site of damage, facilitates access by repair complexes, and promotes the use of the error-free repair pathway of homologous recombination-mediated repair rather than the error-prone process of nonhomologous end joining [118]. BRCA2 affects the choice between the two homologous recombination pathways in favor of the error-free one, by interacting with RAD51 [118]. When the wild-type BRCA1 or BRCA2 allele is lost, mutated, or silenced, a high degree of chromosome instability is observed and defective DNA repair may occur, with the consequent accumulation of additional mutations during replication and promotion of carcinogenesis [119].

double-strand DNA binding domain in the C-terminus [113, 116].

adjuvant and neoadjuvant therapies.

12 Biomarker - Indicator of Abnormal Physiological Process

5.1. BRCA1 and BRCA2

become clinically apparent.

cations.

Among BRCA1 mutation carriers, the estimated breast cancer risk is about 65%, the estimated risk of contralateral breast cancer occurrence results 60%, the cumulative risk of ovarian cancer varies between 27 and 45%, and there is also a significantly increased risk of fallopian tube, uterine and cervical cancer, as well as of male breast cancer, stomach, pancreatic, colon and testicular cancer [123–126].

Among BRCA2 mutation carriers, the estimated lifetime breast cancer risk ranges between 45 and 84%, and that of ovarian cancer between 10 and 20% [123, 127]. There is an increased male breast cancer risk of about 6%, as well as an increased risk of prostate, pancreatic, stomach, gallbladder and bile duct cancers, and malignant melanoma [122, 128].

More than 500 coding region sequence variations have been detected in BRCA1 and 250 in BRCA2. Most unequivocally confirmed mutations reported to date are truncating mutations, adding little in the way of clues for defining functional regions. Although few mutations have been identified in either gene in sporadic breast cancers, a phenotype termed "BRCAness" exist, in which the BRCA1 and BRCA2 proteins act may be somehow disrupted also in sporadic cancer [129]. Finally, there are also some syndromes characterized by an increased breast cancer susceptibility, which are discussed in the following section.

Although germline mutations in BRCA1 and BRCA2 confer a high risk of breast cancer, a great deal of variability has been observed in cancer risk among individuals, both between and within families, as many environmental or genetic factors can modify the penetrance of BRCA1 and BRCA2 mutations. The Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA), analyzing DNA and clinical data from approximately 10,000 BRCA1 and 5000 BRCA2 mutation carriers, demonstrated the role of a number of gene variants in affecting the penetrance in mutation carriers [130].

The most important prognostic modifiers of BRCA1 and BRCA2 mutation carriers are prophylactic oophorectomy and the use of tamoxifen for chemoprevention, which approximately halve the breast cancer risk and decrease the risk of ovarian cancer by about 95% [131, 132].

In comparison with sporadic breast cancers, those related with high-penetrance susceptibility genes correlate with younger age at diagnosis, more aggressive tumor biological behavior, bilaterality, and the eventual coexistence of other cancers in the same individual or in other individuals of the same family, including ovarian, colon, prostate, pancreatic, and endometrial cancers, as well as sarcomas and male breast cancer [133].

Breast cancers arising in BRCA1 mutation carriers are frequently, although not exclusively, basal-like and ER-negative, probably because BRCA1 plays a crucial role in the transcriptional regulation of ER [129, 134]. On the other hand, breast cancer originated in BRCA2 mutation carriers are typically much more similar to sporadic cases, despite with higher grading, higher frequency of ER-positivity and lower frequency of HER2/neu overexpression [135].

The three multigene tests for breast cancer which are commercially available and currently used in the clinical practice are the Oncotype DX test (Genomic Health, Redwood, CA, USA), the MammaPrint test (Netherlands Cancer Institute™ and Agendia™, Netherland), and the Prosigma one (NanoString Technologies, Seattle, WA, USA). The Oncotype DX test is the most widely used molecular test in the therapeutic decision-making, is strongly predictive for endocrine responsiveness in hormone receptor-positive breast cancers with 0–3 positive nodes, and is recommended by both the National Comprehensive Cancer Network (NCCN) and the

Biomarkers in Breast Cancer

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http://dx.doi.org/10.5772/intechopen.77320

The Oncotype DX is a real-time reverse transcriptase chain reaction (RT-PCR) assay, which measures the expression of a panel of 21 genes in formalin-fixed paraffin-embedded samples, including 16 cancer-related genes (ER, PR, Bcl2, SCUBE2, HER2, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, stromelysin 3, cathepsin L2, GSTM1, CD68, and BAG1) and 5 housekeeping control genes (beta-actin, GAPDH, RPLPO, GUS, and TFRC), to generate a recurrence score to stratify breast cancer patients into three risk groups. The low risk group (score < 18), the intermediate (score 18–30), and the high risk one (score ≥ 31) have a 10-year distant recurrence

The MammaPrint assay is the second most commonly ordered molecular test approved by the US-FDA and measure the expression of 70 genes involved in the cell cycle, invasion, proliferation, angiogenesis, metastasis, and signal transduction, none of which is tested by the Oncotype DX assay. The MammaPrint customized microarray contains a reduced set of 1900 probes suitable for high-throughput processing, and allows the use of less RNA and a short processing time of 5 days. This assay can be applied in both node-positive and node-negative and both hormone-positive and hormone-negative cancers, it is predictive for responsiveness to chemotherapy and prognostic for early distant recurrence within the first 10 years after diagnosis, which results 13 and 56% respectively in the low- and high-risk group [147–149].

The Prosigna test is an assay approved by the US-FDA which measures the expression of 50 target genes and 5 constitutively expressed normalization genes, using a proprietary technology called the "nCounter Dx Analysis System." The assay is highly sensitive and precise and uses 250 ng of RNA from formalin-fixed paraffine-embadded tumor tissue, and generates a risk of recurrence score, which assesses the 10-year risk of distant recurrence for hormone receptor-positive stage I–III breast cancers to be treated with adjuvant endocrine therapy, and correlates to one of the five molecular subtypes previously described [143, 150]. The Prosigna assay results superior to the Oncotype DX test in predicting late distant recurrence after

The authors would like to thank the whole collaborating staff of the Universitât dal Friûl, and

St. Gallen Consensus [7, 145, 146].

5–10 years [9, 151].

Acknowledgements

the support from Ennergi research non-profit association.

rate of respectively 6.8, 14.3, and 30.5% [144].

Breast cancer prognosis in BRCA1 and BRCA2 mutation carriers is still controversial, and if some authors describe a worse prognosis even in classically low-risk node-negative disease [136], other exclude any significant difference if compared to the general breast cancer population [137]. Furthermore, BRCA1 and BRCA2 mutation carriers result to have an increased risk of contralateral breast cancer occurrence [138].

New drugs have been purposed as a promising therapeutic strategy in BRCA defective tumor cells, such as the inhibitors of the poly(adenosine diphosphate-ribose) polymerase-1 (PARP1), which is an enzyme involved in the single-stranded DNA repair that use base excision repair.

Other breast cancer susceptibility genes have been described, which can be divided into three categories in terms of mutation risk and the frequency of mutation. Along with BRCA1 and BRCA2 gene mutation, the first category includes PTEN and TP53 gene mutations, which are classified as high-penetrance, low-frequency predisposition genes, and the occurrence of even one of these mutations can increase the risk of breast cancer to 25% [139, 140]. The second category includes the CHEK2, ATM, PALB and BRIP1 2 genes, which are moderate-penetrance, low-frequency predisposition genes, and lead to an increased risk of cancer of twofold to fourfold [110]. Finally, the third category consists of the FGFR2, MAP3K1,and TGFB1 gene mutations, which are low-penetrance, high-frequency predisposition genes [141].

#### 5.2. Multigene signatures

In the last decades, many genomic and molecular classification have been described with a prognostic intent. The most famous divides breast cancers into the following subtypes: luminal A, luminal B, HER2-enriched, basal-like, and normal-like [142]. Luminal subtypes express high levels of ER, they usually have an indolent clinical course, with a low distant recurrence rate, which anyway persists even up to 15 years after the diagnosis. Luminal B subtypes express fewer ER-related genes, have a higher proliferation rate and may overexpress HER2/neu, so that they usually require to be treated with both hormonal therapy and chemotherapy. HER2 enriched subtype exhibits HER2/neu gene amplification but does not express ER-related genes, they have an aggressive natural clinical course but fortunately respond very well to HER2 targeted therapy. The basal-like or triple-negative subtype does not express ER, PR, and HER2/ neu but expresses basal cytokeratins 5/6 and 17, they have a poor prognosis and a high recurrence rate.

The first molecular signature of breast cancer was determined in 2000 by the expression of a set of genes within the tumor, which were able to predict the clinical outcome [143]. The main limitations of gene signature profiling include difficulties in reproducing the specific gene sets, testing expense, and reporting standardization. However, gene signature cannot be substituted by IHC surrogates which have significant discordances with genetic profiling [144].

The three multigene tests for breast cancer which are commercially available and currently used in the clinical practice are the Oncotype DX test (Genomic Health, Redwood, CA, USA), the MammaPrint test (Netherlands Cancer Institute™ and Agendia™, Netherland), and the Prosigma one (NanoString Technologies, Seattle, WA, USA). The Oncotype DX test is the most widely used molecular test in the therapeutic decision-making, is strongly predictive for endocrine responsiveness in hormone receptor-positive breast cancers with 0–3 positive nodes, and is recommended by both the National Comprehensive Cancer Network (NCCN) and the St. Gallen Consensus [7, 145, 146].

The Oncotype DX is a real-time reverse transcriptase chain reaction (RT-PCR) assay, which measures the expression of a panel of 21 genes in formalin-fixed paraffin-embedded samples, including 16 cancer-related genes (ER, PR, Bcl2, SCUBE2, HER2, GRB7, Ki-67, STK15, survivin, cyclin B1, MYBL2, stromelysin 3, cathepsin L2, GSTM1, CD68, and BAG1) and 5 housekeeping control genes (beta-actin, GAPDH, RPLPO, GUS, and TFRC), to generate a recurrence score to stratify breast cancer patients into three risk groups. The low risk group (score < 18), the intermediate (score 18–30), and the high risk one (score ≥ 31) have a 10-year distant recurrence rate of respectively 6.8, 14.3, and 30.5% [144].

The MammaPrint assay is the second most commonly ordered molecular test approved by the US-FDA and measure the expression of 70 genes involved in the cell cycle, invasion, proliferation, angiogenesis, metastasis, and signal transduction, none of which is tested by the Oncotype DX assay. The MammaPrint customized microarray contains a reduced set of 1900 probes suitable for high-throughput processing, and allows the use of less RNA and a short processing time of 5 days. This assay can be applied in both node-positive and node-negative and both hormone-positive and hormone-negative cancers, it is predictive for responsiveness to chemotherapy and prognostic for early distant recurrence within the first 10 years after diagnosis, which results 13 and 56% respectively in the low- and high-risk group [147–149].

The Prosigna test is an assay approved by the US-FDA which measures the expression of 50 target genes and 5 constitutively expressed normalization genes, using a proprietary technology called the "nCounter Dx Analysis System." The assay is highly sensitive and precise and uses 250 ng of RNA from formalin-fixed paraffine-embadded tumor tissue, and generates a risk of recurrence score, which assesses the 10-year risk of distant recurrence for hormone receptor-positive stage I–III breast cancers to be treated with adjuvant endocrine therapy, and correlates to one of the five molecular subtypes previously described [143, 150]. The Prosigna assay results superior to the Oncotype DX test in predicting late distant recurrence after 5–10 years [9, 151].
