Clin Genet 2014: 86: 62–67 Printed in Singapore. All rights reserved
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12381
Review
Personalized therapy for breast cancer De Abreu F.B., Schwartz G.N., Wells W.A., Tsongalis G.J. Personalized therapy for breast cancer. Clin Genet 2014: 86: 62–67. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. For many years, this disease has been classified according to histopathologic criteria, known as the tumor, node and metastasis (TNM) staging system. Clinical criteria that include immunohistochemical markers, such as the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2), provide a classification of breast cancer and dictates the optimal therapeutic approach for treatment. With genomic techniques, such as real-time reverse transcriptase PCR (RT-PCR), microarrays, next-generation sequencing, and whole-exome sequencing, breast cancer diagnostics is going through a significant evolution. Genomic and transcriptomic technologies make the analysis of gene expression signatures and mutation status possible so that tumors may now be classified more accurately with respect to diagnosis and prognosis. The -omic era has also made the possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated either into clinical testing or clinical trials. Together, clinical and molecular criteria can contribute to a more personalized management of the breast cancer patient. This article will present the progress made in the diagnosis and management of breast cancer using molecular information provided by genomic and transcriptomic technologies. Conflict of interest
The authors have no conflicts of interest to declare.
Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. Traditionally, breast cancer has been classified according to histopathologic criteria, including grade, tumor size, and lymph node involvement, also known as the tumor, node and metastasis (TNM) staging system. In addition, clinical criteria including immunohistochemical markers [i.e. estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)] (Fig. 1), are routinely used in diagnostic laboratories to provide a classification of breast cancer and to help determine the optimal approach for treatment. In the past decade, significant advances in molecular biology technologies, such as microarrays, next-generation sequencing, and whole-exome sequencing, have allowed researchers to better understand tumor cell biology to identify complex genomic abnormalities
62
F.B. De Abreua , G.N. Schwartzb,c , W.A. Wellsa and G.J. Tsongalisa,c a Department
of Pathology , b Department of Hematology-Oncology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA , and c Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon NH, USA
Key words: breast cancer – molecular oncology – personalized medicine – pharmacogenomics Corresponding author: Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. Tel.: 603-650-5498; fax: 603-650-4845; e-mail: [email protected] Received 31 January 2014, revised and accepted for publication 1 March 2014
(i.e. gene mutation, copy number aberrations, methylation and translocations), and to identify biomarkers involved in multiple signaling pathways that can improve general clinical practice contributing to a personalized prognostic and predictive approach to management (1). A personalized prognostic and predictive approach consists of increasing therapeutic efficacy by targeting the genomic aberrations that drive tumor behavior whereas at the same time decreasing inadvertent toxicity due to altered drug metabolism determined by the patients’ genetic background (2). Molecular profiling of tumors has helped to individualize the diagnosis and treatment of breast cancer. This article will provide an overview of (i) the molecular subtypes of breast cancer, (ii) targeted therapy for breast cancer (based on traditional clinical classification), (iii) new biomarkers identified in molecular studies that are
Personalized therapy for breast cancer (a)
(b)
(c)
Fig. 1. Clinical classification of breast cancer biomarkers [estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)] using current methods: immunohistochemistry (IHC) (a–c). (a) ER-positive strong intensity (×20). (b) PR-positive moderate intensity (×20). (c) HER-2 overexpression, score 3+.
in phase I–III clinical trials, and (iv) the relevance of using commercially available molecular signatures for predicting outcomes.
Molecular profile of breast cancer
In 2000, Perou et al. (3) designed the first breast cancer molecular profile that classified breast cancer into four subtypes: luminal, HER2 enriched, basal-like and normal breast-like, based on gene expression patterns using hierarchical clustering, which groups genes and samples according to similarity in their patterns of gene expression. In 2006, an expansion of this study in a larger group of patients showed that the luminal group could
be divided into two categories: luminal A and luminal B (4). Furthermore, in 2007, the same group of researchers reported a new molecular subtype: the claudin-low type (5). Table 1 summarizes the characteristics of the molecular subtypes. According to the literature, molecular classification of breast cancer provides a link between the molecular biology of breast cancer and the behavior of cancer cells in the corresponding subtypes (6). It also provides more accurate information about the molecular profile of the tumor than the clinical or phenotypic classification (6–8). However, true molecular profiling has not yet reached clinical implementation as a routine aspect of patient management.
Table 1. Clinical and molecular criteria based on molecular subtypes for breast cancer Molecular subtypes Clinical criteria
Luminal A
Histologic grade Low IHC markers ER+; PR+; HER2−
Luminal B
HER2-positive
Basal-like (or TNBC)
Claudin-low (or TNBC)
High ER+; PR+/−; HER2+/−
High ER−; PR−; HER2+
High High ER−; PR−; HER2− ER−; PR−; HER2−
Molecular criteria Frequency Prognosis Genetic profile
50–60% 10–20% Good Intermediate/poor ER-related genes and High proliferation low proliferation genes genes
15–20% Poor HER2-related genes and high proliferation genes
10–20% Poor CKs, P-cadherin, CAV1/2, CD44, KIT
Genetic instabilities
Low Ki67; CK8/18+; EGFR+; high Ki67, PIK3CA and TP53 PIK3CA and TP53 PIK3CA and mutations mutations TP53 mutations SERMs (tamoxifen) and AIs HER2 target therapy
EGFR+; CK5/6+, high VEGF expression −
Treatment
New treatment PI3K/AKT/mTOR pathway inhibitors, targets on clinical CDK4/6 inhibitors, histone deacetylase inhibitors trials
PI3K/AKT/mTOR pathway inhibitors, neratinib, HSP90
12–14% Poor Low cell–cell junction genes and high immune response genes −
−
PARP-1 inhibitors, − EGFR inhibitor, PI3K/AKT/mTOR pathway inhibitors,
AI, hormonal aromatase inhibitor; CK, cytokines; EGFR, epidermal growth factor receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; PARP-1, poly [ADP-ribose] polymerase 1; PR, progesterone receptor; SERMs, selective estrogen receptor modulators; TNBC, triple negative breast cancer; VEGF, vascular endothelial growth factor.
63
De Abreu et al. Targeted therapy for breast cancer
The advances in genomics and transcriptomics have provided a better understanding of tumor cell biology and also made possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated into clinical testing, contributing to a personalized prognostic and predictive approach to management. The ER, PR and HER2 are well-established biomarkers which are evaluated at diagnosis and characterize the three main immunophenotypes of breast cancer: ER-positive, HER2-positive and ERnegative/PR-negative/HER2-negative. These phenotypes are responsible for directing the selection of the optimal therapeutic approaches to treatment (i.e. endocrine, anti-HER2 and chemotherapy), in determining tumor response to treatments and development of resistance to therapies. ER-positive breast cancer
ER, a member of the nuclear transcription receptor superfamily, is activated by steroid hormones, such as estrogen. Estrogen and its receptors are involved in several processes, including cellular proliferation, inhibition of apoptosis, invasion, and angiogenesis (9). ER has two isoforms, ER-α and ER-β, both expressed in normal mammary gland loping breast cancer tissue,
but ER-α is directly involved in pathological processes, including breast cancer. ERα-positive tumors are well-differentiated, less aggressive, have a better prognosis and represent the majority of luminal tumors: luminal A (with greater expression of estrogen-regulated genes and luminal epithelial markers) and a smaller luminal B fraction (with greater expression of proliferative markers) (10). ERα-positive expression is considered one of the most important biomarkers in breast cancer and represents the principal qualifier for endocrine therapy. Endocrine therapy is divided into two classes of drugs: selective estrogen receptor modulators (SERMs) (tamoxifen, raloxifene, and toremifene) and aromatase inhibitors (AIs) (non-steroidal: letrozole and anastrozole, steroidal: exemestane) (Fig. 2). Selective estrogen receptor modulators
SERMs are drugs that target the ER and suppress the estrogen signaling in women with breast cancer. The US Food and Drug Administration (FDA) has approved three SERMs, tamoxifen, toremifene, and raloxifene, either for treatment of women with the disease or for prevention in women with high risk of developing the disease. Tamoxifen is a SERM that blocks steroid mechanisms preventing cellular replication and proliferation and it has been routinely used to treat all stages of breast cancer. Phase III breast cancer prevention trials have
Fig. 2. Therapies targeting estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-positive breast cancer.
64
Personalized therapy for breast cancer shown the efficacy of tamoxifen in women at high risk of developing breast cancer. For example, the National Surgical Adjuvant Breast and Bowel Project (NSABP) Breast Cancer Prevention Trial (BCPT) P-1 tested the effects of 5 years of tamoxifen treatment in pre- and post-menopausal women at high risk of invasive breast cancer. It showed that tamoxifen reduced the incidence of invasive breast cancer and ER-positive breast cancers by 49% and 69%, respectively (11). In 2007, the International Breast Cancer Intervention Study I (IBIS-I) trial tested women at high risk treated with tamoxifen for 5 years. It showed a 48% reduction in occurrence of ER-positive breast cancer and a 31% reduction in invasive ER-positive breast cancer after long-term follow-up (12). Recently, the Adjuvant Tamoxifen Longer Against Shorter (ATLAS) trial showed that tamoxifen reduces both breast cancer recurrence and the development of a new contralateral breast cancer by approximately 40–50% in women with early breast cancer (13). Tamoxifen also has activity in ductal carcinoma in situ (DCIS) and in metastatic disease. The NSABP-B24 trial verified the effect of tamoxifen vs placebo in 1800 women undergoing breast surgery and radiation for DCIS for 5 years. After a 7-year follow-up, it showed a 48% reduction in invasive breast cancer patients receiving tamoxifen (14, 15). Raloxifene is a SERM that has anti-estrogen activity and has been used for preventive treatment of post-menopausal women with osteoporosis and/or in those patients at high risk of invasive breast cancer. The Multiple Outcomes of Raloxifene Evaluation (MORE) trial tested the effect of raloxifene on bone fractures in post-menopausal women with osteoporosis (16). It showed a 76% reduction in invasive breast cancer incidence and a 90% reduction in ER-positive breast cancer incidence after 3 years of treatment with raloxifene. In 2004, the Continued Outcomes of Raloxifene Evaluation (CORE) trial confirmed that long-term treatment with raloxifene reduces the incidence of invasive ER-positive breast cancer (17). Recently, the Raloxifene Use for the Heart (RUTH) trial observed the effect of 5 years of raloxifene on the incidence of coronary events and invasive breast cancer (18). It showed a reduction in the rate of invasive breast cancer and ER-positive breast cancer by 44% and 55%, respectively. The NSABP Study of Tamoxifen and Raloxifene (STAR) trial was developed to compare the effectiveness of 5 years of treatment with these two drugs in decreasing the risk of breast cancer in high-risk post-menopausal women (19). Vogel et al. (19) reported that the two drugs were equally effective in preventing invasive breast cancer but patients treated with raloxifene reported fewer side effects, including fewer cases of blood clots and uterine cancer. However, the STAR trial did not have a placebo group to confirm the absolute activity of either drug from this study. Lasofoxifene is another SERM that does not yet have FDA-approval for breast cancer indications, which include either treatment or prevention. Recently, the Postmenopausal Evaluation and Risk Reduction with
Lasofoxifene (PEARL) trial reported an 81% reduction in ER-positive breast cancer incidence in patients treated with lasofoxifene for 5 years (20). This drug was associated with decreased toxicity than that usually seen with tamoxifen and raloxifene. Aromatase inhibitor
AIs, such as letrozole, anastrozole, and exemestane, block the biosynthesis of estrogen from androgens through the inhibition of the aromatase enzyme, resulting in severe reductions in the circulating estrogen levels of serum, tissue, and tumor cells. AIs are a more effective treatment strategy in post-menopausal women with ER-positive breast cancer than tamoxifen (21). Multiple clinical trials demonstrated an improvement in response rate and progression-free survival in women with ER-positive metastatic breast cancer, and a reduction in the risk of local, regional, and distant recurrence in women with ER-positive early stage breast cancer randomized to treatment with an AI compared to women randomized to treatment with tamoxifen. Several clinical trials demonstrated improved efficacy in the prevention of a second primary breast cancer following treatment with AIs as compared to tamoxifen (22, 23), and two studies have demonstrated the efficacy of using an AI for primary breast cancer prevention (24, 25). HER2-positive breast cancer
HER2 belongs to the HER family of tyrosine kinases, which includes EGFR (HER1), HER3, and HER4. HER2 can regulate cell proliferation, survival, and other processes important for carcinogenesis. The activation of HER2, which occurs through gene amplification leading to receptor protein overexpression, is identified in 15–20% of all breast cancers and is associated with a more aggressive phenotype. Currently, HER2 gene amplification is used as a pharmacogenomics test to identify breast cancer patients who may benefit from treatment with HER2-targeted agents, such as trastuzumab (or herceptin), lapatinib, pertuzumab, and trastuzumab emtansine (T-DM1) (Fig. 2). Treatment targeting HER2 using different molecular mechanisms (HER2 and EGFR inhibitors) promotes inhibition of tumor growth and induces apoptosis. Trastuzumab
Trastuzumab is a monoclonal antibody that targets the HER2 extracellular domain, blocking its activation and it has been approved for both metastatic and adjuvant therapy in HER2-positive breast cancer. Unfortunately, not all HER2-positive patients respond to trastuzumab therapy. Furthermore, metastatic tumors that initially respond to treatment often develop resistance. A combined HER2 targeting strategy with trastuzumab and lapatinib showed efficacy in trastuzumab-resistant metastatic HER2-positive breast cancer (26). HER2 is amplified in approximately 10% of ER-positive breast cancer. ER-positive/HER2-positive tumors have a higher risk of relapse on adjuvant
65
De Abreu et al. endocrine therapy than ER-positive/HER2-negative tumors (27). However, a significant reduction in risk of relapse was observed when ER-positive/HER2-positive patients were treated with trastuzumab. The combination of hormonal therapy and trastuzumab is considered standard of care for these patients. Lapatinib, pertuzumab, and trastuzumab emtansine
Lapatinib is a small molecule inhibitor of the kinase activity of HER2 and EGFR. It has been FDA-approved for HER2-positive breast cancer patients. Pertuzumab is monoclonal antibody that also binds to extracellular domain of HER2 inhibiting dimerization of HER2 with other HER family members. This drug was FDA-approved in combination with docetaxel and trastuzumab for HER2-positive metastatic disease, and in combination with neoadjuvant chemotherapy and trastuzumab for patients with locally advanced HER2-positive breast cancer. T-DM1 is an antibodydrug conjugate in which trastuzumab is linked to the microtubule-inhibitory agent mertansine (DM1) (28). T-DM1 is FDA-approved for the treatment of patients with HER2-positive metastatic breast cancer previously treated with trastuzumab and taxane, separately or in combination. Triple negative breast cancer
Triple negative breast cancers (TNBCs) represent 15–20% of all breast cancers and are characterized by the lack of ER, PR and HER2 expression. TNBCs are more aggressive and have a worse clinical outcome. Unlike ER-positive and HER2-positive breast cancer, there is no approved targeted therapy for TNBC. However, the classification of breast tumors into subtypes based on specific gene expression profiles may contribute to the development of targeted agents. Lehmann et al. (29) analyzed gene expression of 587 TNBC cases from 21 breast cancer data sets and identified 6 TNBC subtypes, basal-like (BL1 and BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptor (LAR). According to the authors, BL1 and BL2 subtypes include genes involved in cell cycle and growth factor pathways, respectively; IM involves genes responsible for immune cell processes; M and MSL have genes responsible for cell motility and cellular differentiation; and the LAR subtype is characterized by androgen receptor signaling. The difference between M and MSL is that MSL expresses low levels of proliferative genes. The identification of these subtypes can select driver biomarkers contributing to drug discovery and designing of clinical trials. Clinical trials
Advances in genomic and transcriptomic technologies has allowed for a better understanding of tumor cell biology and, consequently, to the identification of novel biomarkers involved in multiple signaling pathways that
66
can improve general clinical practice and contribute to a personalized prognostic and predictive approach to clinical subgroups of breast cancer (Fig. 2). Recently, several clinical trials are in progress investigating the effect of PI3K/AKT/mTOR inhibitors, CDK4/6 inhibitors, histone deacetylases (HDACs) inhibitors, and poly-adenosine diphosphate-ribose polymerase (PARPs) inhibitors, involved either in cell proliferation, metabolism, motility, angiogenesis, apoptosis or DNA damage repair. More information about each pathway inhibitors and clinical trials in patients with primary or metastatic breast cancer is available in Appendix S1, Supporting Information. Gene expression profiles used in clinical decision making
Currently, molecular diagnostic assays, such as MammaPrint® (Agendia, Amsterdam, The Netherlands), Oncotype DX® (Genomic Health, Inc., Redwood City, CA) and the Genomic Grade Index (GGI), using RT-PCR or microarray technology, identify gene signatures to predict response to therapy. MammaPrint is a microarray gene expression profiling test that analyzes 70 critical genes, involved in cell cycle, invasion, metastasis, angiogenesis, and signal transduction, and classifies patients into two distinct groups, low risk or high risk of distant recurrence. MammaPrint was developed by a group of researchers who identified a prognostic signature using a microarray platform in node-negative breast cancer patients under the age of 55 (30) and validated in node-negative and node-positive tumors, as well as treated and untreated patients, and proved to be a robust predictor for distant metastatic-free survival, independent of adjuvant treatment, tumor size, histological grade, and age (31). A second validation was performed in node-negative T1-2 breast tumors not treated with chemotherapy and compared to traditional clinical factors included in the Adjuvant Online software. Oncotype DX is a 21-gene expression assay to provide a recurrence score (RS) as a prognostic indicator. The RS predicts the probability of distant recurrence in node-negative ER-positive patients treated with tamoxifen (32). The assay identifies expression of 21 genes (5 reference genes and 16 genes associated with breast cancer), selected from a set of 250 genes previously studied by the NSABP clinical studies, and classifies patients in three groups: high, intermediate and low risk. The RS was shown to correlate with distant recurrence, relapse-free interval, and overall survival, independent of age and tumor size. The GGI signature was developed to reclassify patients with histologic grade 2 tumors, which is informative for clinical decision making. Sotiriou et al. (33) analyzed microarray data from 189 invasive breast cancers and identified 97 genes associated with histologic grade, most of them involved in cell cycle regulation and proliferation. These genes were differentially expressed between high-grade and low-grade breast tumors. The intermediate grade tumors showed an expression pattern similar to either high-grade or low-grade cases. The GGI
Personalized therapy for breast cancer may increase the accuracy of tumor grading and improve treatment decisions. Conclusion
Advances in genomic and transcriptomic assessment of breast cancers have provided a better understanding of disease processes and have proven more beneficial than traditional methods of tumor evaluation in the laboratory for more specific patient management. New biomarkers and reclassification of breast cancers have also led to the development of novel therapies and treatment strategies for the breast cancer patient. Together, this new information can contribute to a more personalized management of the breast cancer patient. Supporting Information
14.
15.
16.
17.
18.
19.
The following Supporting information is available for this article: Appendix S1. Inhibitors of pathways involved in breast cancer.
20.
Additional Supporting information may be found in the online version of this article.
21.
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12381
Review
Personalized therapy for breast cancer De Abreu F.B., Schwartz G.N., Wells W.A., Tsongalis G.J. Personalized therapy for breast cancer. Clin Genet 2014: 86: 62–67. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. For many years, this disease has been classified according to histopathologic criteria, known as the tumor, node and metastasis (TNM) staging system. Clinical criteria that include immunohistochemical markers, such as the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2), provide a classification of breast cancer and dictates the optimal therapeutic approach for treatment. With genomic techniques, such as real-time reverse transcriptase PCR (RT-PCR), microarrays, next-generation sequencing, and whole-exome sequencing, breast cancer diagnostics is going through a significant evolution. Genomic and transcriptomic technologies make the analysis of gene expression signatures and mutation status possible so that tumors may now be classified more accurately with respect to diagnosis and prognosis. The -omic era has also made the possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated either into clinical testing or clinical trials. Together, clinical and molecular criteria can contribute to a more personalized management of the breast cancer patient. This article will present the progress made in the diagnosis and management of breast cancer using molecular information provided by genomic and transcriptomic technologies. Conflict of interest
The authors have no conflicts of interest to declare.
Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. Traditionally, breast cancer has been classified according to histopathologic criteria, including grade, tumor size, and lymph node involvement, also known as the tumor, node and metastasis (TNM) staging system. In addition, clinical criteria including immunohistochemical markers [i.e. estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)] (Fig. 1), are routinely used in diagnostic laboratories to provide a classification of breast cancer and to help determine the optimal approach for treatment. In the past decade, significant advances in molecular biology technologies, such as microarrays, next-generation sequencing, and whole-exome sequencing, have allowed researchers to better understand tumor cell biology to identify complex genomic abnormalities
62
F.B. De Abreua , G.N. Schwartzb,c , W.A. Wellsa and G.J. Tsongalisa,c a Department
of Pathology , b Department of Hematology-Oncology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA , and c Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon NH, USA
Key words: breast cancer – molecular oncology – personalized medicine – pharmacogenomics Corresponding author: Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. Tel.: 603-650-5498; fax: 603-650-4845; e-mail: [email protected] Received 31 January 2014, revised and accepted for publication 1 March 2014
(i.e. gene mutation, copy number aberrations, methylation and translocations), and to identify biomarkers involved in multiple signaling pathways that can improve general clinical practice contributing to a personalized prognostic and predictive approach to management (1). A personalized prognostic and predictive approach consists of increasing therapeutic efficacy by targeting the genomic aberrations that drive tumor behavior whereas at the same time decreasing inadvertent toxicity due to altered drug metabolism determined by the patients’ genetic background (2). Molecular profiling of tumors has helped to individualize the diagnosis and treatment of breast cancer. This article will provide an overview of (i) the molecular subtypes of breast cancer, (ii) targeted therapy for breast cancer (based on traditional clinical classification), (iii) new biomarkers identified in molecular studies that are
Personalized therapy for breast cancer (a)
(b)
(c)
Fig. 1. Clinical classification of breast cancer biomarkers [estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)] using current methods: immunohistochemistry (IHC) (a–c). (a) ER-positive strong intensity (×20). (b) PR-positive moderate intensity (×20). (c) HER-2 overexpression, score 3+.
in phase I–III clinical trials, and (iv) the relevance of using commercially available molecular signatures for predicting outcomes.
Molecular profile of breast cancer
In 2000, Perou et al. (3) designed the first breast cancer molecular profile that classified breast cancer into four subtypes: luminal, HER2 enriched, basal-like and normal breast-like, based on gene expression patterns using hierarchical clustering, which groups genes and samples according to similarity in their patterns of gene expression. In 2006, an expansion of this study in a larger group of patients showed that the luminal group could
be divided into two categories: luminal A and luminal B (4). Furthermore, in 2007, the same group of researchers reported a new molecular subtype: the claudin-low type (5). Table 1 summarizes the characteristics of the molecular subtypes. According to the literature, molecular classification of breast cancer provides a link between the molecular biology of breast cancer and the behavior of cancer cells in the corresponding subtypes (6). It also provides more accurate information about the molecular profile of the tumor than the clinical or phenotypic classification (6–8). However, true molecular profiling has not yet reached clinical implementation as a routine aspect of patient management.
Table 1. Clinical and molecular criteria based on molecular subtypes for breast cancer Molecular subtypes Clinical criteria
Luminal A
Histologic grade Low IHC markers ER+; PR+; HER2−
Luminal B
HER2-positive
Basal-like (or TNBC)
Claudin-low (or TNBC)
High ER+; PR+/−; HER2+/−
High ER−; PR−; HER2+
High High ER−; PR−; HER2− ER−; PR−; HER2−
Molecular criteria Frequency Prognosis Genetic profile
50–60% 10–20% Good Intermediate/poor ER-related genes and High proliferation low proliferation genes genes
15–20% Poor HER2-related genes and high proliferation genes
10–20% Poor CKs, P-cadherin, CAV1/2, CD44, KIT
Genetic instabilities
Low Ki67; CK8/18+; EGFR+; high Ki67, PIK3CA and TP53 PIK3CA and TP53 PIK3CA and mutations mutations TP53 mutations SERMs (tamoxifen) and AIs HER2 target therapy
EGFR+; CK5/6+, high VEGF expression −
Treatment
New treatment PI3K/AKT/mTOR pathway inhibitors, targets on clinical CDK4/6 inhibitors, histone deacetylase inhibitors trials
PI3K/AKT/mTOR pathway inhibitors, neratinib, HSP90
12–14% Poor Low cell–cell junction genes and high immune response genes −
−
PARP-1 inhibitors, − EGFR inhibitor, PI3K/AKT/mTOR pathway inhibitors,
AI, hormonal aromatase inhibitor; CK, cytokines; EGFR, epidermal growth factor receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; PARP-1, poly [ADP-ribose] polymerase 1; PR, progesterone receptor; SERMs, selective estrogen receptor modulators; TNBC, triple negative breast cancer; VEGF, vascular endothelial growth factor.
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De Abreu et al. Targeted therapy for breast cancer
The advances in genomics and transcriptomics have provided a better understanding of tumor cell biology and also made possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated into clinical testing, contributing to a personalized prognostic and predictive approach to management. The ER, PR and HER2 are well-established biomarkers which are evaluated at diagnosis and characterize the three main immunophenotypes of breast cancer: ER-positive, HER2-positive and ERnegative/PR-negative/HER2-negative. These phenotypes are responsible for directing the selection of the optimal therapeutic approaches to treatment (i.e. endocrine, anti-HER2 and chemotherapy), in determining tumor response to treatments and development of resistance to therapies. ER-positive breast cancer
ER, a member of the nuclear transcription receptor superfamily, is activated by steroid hormones, such as estrogen. Estrogen and its receptors are involved in several processes, including cellular proliferation, inhibition of apoptosis, invasion, and angiogenesis (9). ER has two isoforms, ER-α and ER-β, both expressed in normal mammary gland loping breast cancer tissue,
but ER-α is directly involved in pathological processes, including breast cancer. ERα-positive tumors are well-differentiated, less aggressive, have a better prognosis and represent the majority of luminal tumors: luminal A (with greater expression of estrogen-regulated genes and luminal epithelial markers) and a smaller luminal B fraction (with greater expression of proliferative markers) (10). ERα-positive expression is considered one of the most important biomarkers in breast cancer and represents the principal qualifier for endocrine therapy. Endocrine therapy is divided into two classes of drugs: selective estrogen receptor modulators (SERMs) (tamoxifen, raloxifene, and toremifene) and aromatase inhibitors (AIs) (non-steroidal: letrozole and anastrozole, steroidal: exemestane) (Fig. 2). Selective estrogen receptor modulators
SERMs are drugs that target the ER and suppress the estrogen signaling in women with breast cancer. The US Food and Drug Administration (FDA) has approved three SERMs, tamoxifen, toremifene, and raloxifene, either for treatment of women with the disease or for prevention in women with high risk of developing the disease. Tamoxifen is a SERM that blocks steroid mechanisms preventing cellular replication and proliferation and it has been routinely used to treat all stages of breast cancer. Phase III breast cancer prevention trials have
Fig. 2. Therapies targeting estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-positive breast cancer.
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Personalized therapy for breast cancer shown the efficacy of tamoxifen in women at high risk of developing breast cancer. For example, the National Surgical Adjuvant Breast and Bowel Project (NSABP) Breast Cancer Prevention Trial (BCPT) P-1 tested the effects of 5 years of tamoxifen treatment in pre- and post-menopausal women at high risk of invasive breast cancer. It showed that tamoxifen reduced the incidence of invasive breast cancer and ER-positive breast cancers by 49% and 69%, respectively (11). In 2007, the International Breast Cancer Intervention Study I (IBIS-I) trial tested women at high risk treated with tamoxifen for 5 years. It showed a 48% reduction in occurrence of ER-positive breast cancer and a 31% reduction in invasive ER-positive breast cancer after long-term follow-up (12). Recently, the Adjuvant Tamoxifen Longer Against Shorter (ATLAS) trial showed that tamoxifen reduces both breast cancer recurrence and the development of a new contralateral breast cancer by approximately 40–50% in women with early breast cancer (13). Tamoxifen also has activity in ductal carcinoma in situ (DCIS) and in metastatic disease. The NSABP-B24 trial verified the effect of tamoxifen vs placebo in 1800 women undergoing breast surgery and radiation for DCIS for 5 years. After a 7-year follow-up, it showed a 48% reduction in invasive breast cancer patients receiving tamoxifen (14, 15). Raloxifene is a SERM that has anti-estrogen activity and has been used for preventive treatment of post-menopausal women with osteoporosis and/or in those patients at high risk of invasive breast cancer. The Multiple Outcomes of Raloxifene Evaluation (MORE) trial tested the effect of raloxifene on bone fractures in post-menopausal women with osteoporosis (16). It showed a 76% reduction in invasive breast cancer incidence and a 90% reduction in ER-positive breast cancer incidence after 3 years of treatment with raloxifene. In 2004, the Continued Outcomes of Raloxifene Evaluation (CORE) trial confirmed that long-term treatment with raloxifene reduces the incidence of invasive ER-positive breast cancer (17). Recently, the Raloxifene Use for the Heart (RUTH) trial observed the effect of 5 years of raloxifene on the incidence of coronary events and invasive breast cancer (18). It showed a reduction in the rate of invasive breast cancer and ER-positive breast cancer by 44% and 55%, respectively. The NSABP Study of Tamoxifen and Raloxifene (STAR) trial was developed to compare the effectiveness of 5 years of treatment with these two drugs in decreasing the risk of breast cancer in high-risk post-menopausal women (19). Vogel et al. (19) reported that the two drugs were equally effective in preventing invasive breast cancer but patients treated with raloxifene reported fewer side effects, including fewer cases of blood clots and uterine cancer. However, the STAR trial did not have a placebo group to confirm the absolute activity of either drug from this study. Lasofoxifene is another SERM that does not yet have FDA-approval for breast cancer indications, which include either treatment or prevention. Recently, the Postmenopausal Evaluation and Risk Reduction with
Lasofoxifene (PEARL) trial reported an 81% reduction in ER-positive breast cancer incidence in patients treated with lasofoxifene for 5 years (20). This drug was associated with decreased toxicity than that usually seen with tamoxifen and raloxifene. Aromatase inhibitor
AIs, such as letrozole, anastrozole, and exemestane, block the biosynthesis of estrogen from androgens through the inhibition of the aromatase enzyme, resulting in severe reductions in the circulating estrogen levels of serum, tissue, and tumor cells. AIs are a more effective treatment strategy in post-menopausal women with ER-positive breast cancer than tamoxifen (21). Multiple clinical trials demonstrated an improvement in response rate and progression-free survival in women with ER-positive metastatic breast cancer, and a reduction in the risk of local, regional, and distant recurrence in women with ER-positive early stage breast cancer randomized to treatment with an AI compared to women randomized to treatment with tamoxifen. Several clinical trials demonstrated improved efficacy in the prevention of a second primary breast cancer following treatment with AIs as compared to tamoxifen (22, 23), and two studies have demonstrated the efficacy of using an AI for primary breast cancer prevention (24, 25). HER2-positive breast cancer
HER2 belongs to the HER family of tyrosine kinases, which includes EGFR (HER1), HER3, and HER4. HER2 can regulate cell proliferation, survival, and other processes important for carcinogenesis. The activation of HER2, which occurs through gene amplification leading to receptor protein overexpression, is identified in 15–20% of all breast cancers and is associated with a more aggressive phenotype. Currently, HER2 gene amplification is used as a pharmacogenomics test to identify breast cancer patients who may benefit from treatment with HER2-targeted agents, such as trastuzumab (or herceptin), lapatinib, pertuzumab, and trastuzumab emtansine (T-DM1) (Fig. 2). Treatment targeting HER2 using different molecular mechanisms (HER2 and EGFR inhibitors) promotes inhibition of tumor growth and induces apoptosis. Trastuzumab
Trastuzumab is a monoclonal antibody that targets the HER2 extracellular domain, blocking its activation and it has been approved for both metastatic and adjuvant therapy in HER2-positive breast cancer. Unfortunately, not all HER2-positive patients respond to trastuzumab therapy. Furthermore, metastatic tumors that initially respond to treatment often develop resistance. A combined HER2 targeting strategy with trastuzumab and lapatinib showed efficacy in trastuzumab-resistant metastatic HER2-positive breast cancer (26). HER2 is amplified in approximately 10% of ER-positive breast cancer. ER-positive/HER2-positive tumors have a higher risk of relapse on adjuvant
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De Abreu et al. endocrine therapy than ER-positive/HER2-negative tumors (27). However, a significant reduction in risk of relapse was observed when ER-positive/HER2-positive patients were treated with trastuzumab. The combination of hormonal therapy and trastuzumab is considered standard of care for these patients. Lapatinib, pertuzumab, and trastuzumab emtansine
Lapatinib is a small molecule inhibitor of the kinase activity of HER2 and EGFR. It has been FDA-approved for HER2-positive breast cancer patients. Pertuzumab is monoclonal antibody that also binds to extracellular domain of HER2 inhibiting dimerization of HER2 with other HER family members. This drug was FDA-approved in combination with docetaxel and trastuzumab for HER2-positive metastatic disease, and in combination with neoadjuvant chemotherapy and trastuzumab for patients with locally advanced HER2-positive breast cancer. T-DM1 is an antibodydrug conjugate in which trastuzumab is linked to the microtubule-inhibitory agent mertansine (DM1) (28). T-DM1 is FDA-approved for the treatment of patients with HER2-positive metastatic breast cancer previously treated with trastuzumab and taxane, separately or in combination. Triple negative breast cancer
Triple negative breast cancers (TNBCs) represent 15–20% of all breast cancers and are characterized by the lack of ER, PR and HER2 expression. TNBCs are more aggressive and have a worse clinical outcome. Unlike ER-positive and HER2-positive breast cancer, there is no approved targeted therapy for TNBC. However, the classification of breast tumors into subtypes based on specific gene expression profiles may contribute to the development of targeted agents. Lehmann et al. (29) analyzed gene expression of 587 TNBC cases from 21 breast cancer data sets and identified 6 TNBC subtypes, basal-like (BL1 and BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptor (LAR). According to the authors, BL1 and BL2 subtypes include genes involved in cell cycle and growth factor pathways, respectively; IM involves genes responsible for immune cell processes; M and MSL have genes responsible for cell motility and cellular differentiation; and the LAR subtype is characterized by androgen receptor signaling. The difference between M and MSL is that MSL expresses low levels of proliferative genes. The identification of these subtypes can select driver biomarkers contributing to drug discovery and designing of clinical trials. Clinical trials
Advances in genomic and transcriptomic technologies has allowed for a better understanding of tumor cell biology and, consequently, to the identification of novel biomarkers involved in multiple signaling pathways that
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can improve general clinical practice and contribute to a personalized prognostic and predictive approach to clinical subgroups of breast cancer (Fig. 2). Recently, several clinical trials are in progress investigating the effect of PI3K/AKT/mTOR inhibitors, CDK4/6 inhibitors, histone deacetylases (HDACs) inhibitors, and poly-adenosine diphosphate-ribose polymerase (PARPs) inhibitors, involved either in cell proliferation, metabolism, motility, angiogenesis, apoptosis or DNA damage repair. More information about each pathway inhibitors and clinical trials in patients with primary or metastatic breast cancer is available in Appendix S1, Supporting Information. Gene expression profiles used in clinical decision making
Currently, molecular diagnostic assays, such as MammaPrint® (Agendia, Amsterdam, The Netherlands), Oncotype DX® (Genomic Health, Inc., Redwood City, CA) and the Genomic Grade Index (GGI), using RT-PCR or microarray technology, identify gene signatures to predict response to therapy. MammaPrint is a microarray gene expression profiling test that analyzes 70 critical genes, involved in cell cycle, invasion, metastasis, angiogenesis, and signal transduction, and classifies patients into two distinct groups, low risk or high risk of distant recurrence. MammaPrint was developed by a group of researchers who identified a prognostic signature using a microarray platform in node-negative breast cancer patients under the age of 55 (30) and validated in node-negative and node-positive tumors, as well as treated and untreated patients, and proved to be a robust predictor for distant metastatic-free survival, independent of adjuvant treatment, tumor size, histological grade, and age (31). A second validation was performed in node-negative T1-2 breast tumors not treated with chemotherapy and compared to traditional clinical factors included in the Adjuvant Online software. Oncotype DX is a 21-gene expression assay to provide a recurrence score (RS) as a prognostic indicator. The RS predicts the probability of distant recurrence in node-negative ER-positive patients treated with tamoxifen (32). The assay identifies expression of 21 genes (5 reference genes and 16 genes associated with breast cancer), selected from a set of 250 genes previously studied by the NSABP clinical studies, and classifies patients in three groups: high, intermediate and low risk. The RS was shown to correlate with distant recurrence, relapse-free interval, and overall survival, independent of age and tumor size. The GGI signature was developed to reclassify patients with histologic grade 2 tumors, which is informative for clinical decision making. Sotiriou et al. (33) analyzed microarray data from 189 invasive breast cancers and identified 97 genes associated with histologic grade, most of them involved in cell cycle regulation and proliferation. These genes were differentially expressed between high-grade and low-grade breast tumors. The intermediate grade tumors showed an expression pattern similar to either high-grade or low-grade cases. The GGI
Personalized therapy for breast cancer may increase the accuracy of tumor grading and improve treatment decisions. Conclusion
Advances in genomic and transcriptomic assessment of breast cancers have provided a better understanding of disease processes and have proven more beneficial than traditional methods of tumor evaluation in the laboratory for more specific patient management. New biomarkers and reclassification of breast cancers have also led to the development of novel therapies and treatment strategies for the breast cancer patient. Together, this new information can contribute to a more personalized management of the breast cancer patient. Supporting Information
14.
15.
16.
17.
18.
19.
The following Supporting information is available for this article: Appendix S1. Inhibitors of pathways involved in breast cancer.
20.
Additional Supporting information may be found in the online version of this article.
21.
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