Hormones can be classified generally into 2 broad groups: (a) nonsteroidal (amino acids, peptides, and polypeptides), which usually require cell-membrane localized receptors that regulate second messenger molecules such as cyclic adenosine monophosphate (cAMP) to mediate their action (see Chap. 8, Sec. 8.2), and (b) steroidal, which bind directly to intracellular receptors to mediate their action. Breast and prostate cancer are dependent primarily on estrogen and androgen steroid hormones, respectively, for their growth and viability. Other examples of steroid hormones include glucocorticoids, mineralocorticoids, and progestins such as progesterone.
The bioavailability of steroid hormones at the site of action depends on several factors, including synthesis, transport via the blood, access to target tissue, metabolism in target tissue, and expression of receptors within the target cell.
20.2.1 Synthesis and Metabolism of Estrogens
As indicated in Figure 20–1, all steroids are synthesized from the common precursor, cholesterol. The primary site of synthesis of estrogens (eg, estrone and estradiol) in premenopausal women is the parafollicular region of the ovary. Ovarian steroid synthesis in premenopausal women is cyclical and regulated via the gonad–hypothalamus–pituitary feedback axis, as indicated in Figure 20–2A. Other sites of estrogen biosynthesis include mesenchymal cells in adipose tissue and skin; these tissues become major sources for estrogen synthesis in postmenopausal women where adrenal androgens, in particular androstenedione, are converted to estrone by aromatase cytochrome P450 (Simpson, 2000). Some estrone molecules can then be converted to the more potent estradiol-17β by 17β-hydroxysteroid dehydrogenases (Miettinen et al, 2000). A large amount of estrone is converted by estrone sulfotransferase to estrone sulphate, which has a longer half-life in blood, and therefore can act as an estrogen reservoir, being converted back to estrone by the action of sulfatases (Miettinen et al, 2000). Aromatase activity has been detected in normal human breast tissue and in more than 50% of human breast tumors (Sasano and Harada, 1998). Estrone sulfotransferase, sulfatase and 17β-hydroxysteroid dehydrogenase type 1 have been detected in both normal and malignant human breast tissues (Miettinen et al, 2000) and the relative expression of these enzymes probably regulates the local availability of estrogens to target cells. Estrogen synthesis in postmenopausal women is not cyclical but serum and local tissue levels can differ between individuals because of a variety of environmental and genetic factors such as obesity and genetic variation/polymorphism in steroid metabolizing (biosynthetic and degrading) enzymes (Thompson and Ambrosone, 2000).
The principal mammalian steroid biosynthetic pathways for estradiol, testosterone, and dihydrotestosterone are represented by solid arrows. The enzymes that catalyze the reactions are shown in boxes. Although the main sites for steroid synthesis are the gonads and the adrenals, metabolic interconversions and activations occur in sex hormone target tissues, such as the prostate and breast, as well as other peripheral tissues, such as skin and adipose tissue. Dashed arrows and boxes represent the intratumoral androgen synthesis pathway. Inhibitors of enzymes are represented in blue ovals.
20.2.2 Synthesis and Metabolism of Androgens
The Leydig cells of the testes produce almost 90% of the body's androgens with the remainder being made mainly by the adrenal cortex. As illustrated in Figure 20–2B, the testes make primarily testosterone, whereas the major adrenal androgens are dehydroepiandrosterone (DHEA) and its derived sulfate, which although weak androgens, can be converted in other tissues to testosterone. The principal circulating androgen in man is testosterone (see Fig. 20–1). As with estrogen synthesis in the ovary, testosterone production in the testis is regulated by a negative feedback loop involving luteinizing hormone (LH) and luteinizing hormone-releasing hormone (LHRH) via the gonad–hypothalamus–pituitary feedback axis (see Fig. 20–2B). Although there are diurnal fluctuations of androgen secretion, their production does not follow a regular cyclical pattern. Also, there is no apparent equivalent to the menopause in men, although there is a progressive decrease in testosterone levels with age, which is accompanied by some degree of testicular failure.
The gonadal–hypothalamic–pituitary axis. The pathways and feedback loops that regulate the production of estrogens in females (A) and androgens in males (B) and their target tissues are shown. The procedures and agents used for blocking the synthesis and activity of androgens and estrogens at the various steps in the pathway are highlighted. Note that luteinizing hormone-releasing hormone (LHRH) agonists initially stimulate release of luteinizing hormone (LH) followed by its inhibition. ACTH, adrenocorticotropic hormone; FSH, follicle-stimulating hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone.
There are 2 principal pathways through which testosterone can undergo metabolic conversion to more potent forms (see Fig. 20–1). The first involves the enzyme aromatase, which is present in many tissues (eg, adipose tissue, testis, brain) and which can convert approximately 0.5% of the daily production of testosterone to estradiol. Although this is a small proportion of the total amount, estradiol is a 200-fold more potent inhibitor of gonadotrophins than testosterone. The second major pathway is the conversion of testosterone to the more potent androgen dihydrotestosterone (DHT) by the enzyme 5α-reductase, which is present in many androgen target tissues such as the prostate and skin. There are 2 isoforms of 5α-reductase, each with different kinetic properties and sensitivity to inhibitors, with the type II isoform predominating in human accessory sex tissue (Bruchovsky et al, 1988). The interaction of testosterone and DHT with the androgen receptor is different. Testosterone has a 2-fold lower affinity than DHT for the androgen receptor and is more effective in regulating differentiation. However, the dissociation rate of DHT from the receptor is 5-fold slower than testosterone, and it is a more potent androgen to induce growth and maintenance of the prostate gland (Grino et al, 1990).
20.2.3 Transport of Steroid Hormones in the Blood
As hydrophobic molecules in an aqueous environment, most steroid hormones are transported in the blood bound to proteins, predominantly sex hormone-binding globulin (SHBG) and albumin (as shown in Fig. 20–3). Only approximately 2% is in an unbound form, which is the biologically active fraction. SHBG also plays a role in permitting certain steroid hormones to act without entering the cell: estrogens and androgens bind with high affinity to SHBG, which, in turn, interacts with a specific, high-affinity receptor (SHBG-R) on cell membranes and transduces a signal via a G-protein/cAMP (Kahn et al, 2002). The steroid/SHBG-R/SHBG complex generates messages that have effects on the transcriptional activity of regular, intracellular receptors for steroid hormones. Hence, SHBG not only modulates the amount of ligand available to bind to these steroid receptors, but may also modulate their activity through interaction with the cell membrane. Factors that influence the levels of SHBG and albumin will affect the bioactivity of steroid hormones. For example, SHBG production is stimulated by both estrogens and by the antiestrogen tamoxifen, whereas androgens and progestins have been shown to suppress it. In addition, SHBG can be expressed inside the cytoplasm of prostate cells from de novo synthesis and, through binding to steroids such as DHT, can affect the intracellular concentration of free androgens (Kahn et al, 2002).
Schematic pathways for the molecular action of testosterone and estradiol in hormone target cells. A relatively simplistic overview of the key events in steroid hormone action with the sites at which the hormone signal can be inhibited, are indicated. Although the dynamics of estrogen and androgen action are similar, for full activity testosterone needs to be converted to dihydrotestosterone, which binds to a cytoplasmic form of the androgen receptor and is translocated into the nucleus, whereas estradiol binds directly to its receptor in the cell nucleus. ARE, androgen-responsive element; ERE, estrogen-responsive element; HSP, heat shock protein; SHBG, sex hormone-binding globulin.
20.2.4 Steroid Hormone Receptors
An overview of how steroid hormones regulate growth and differentiation of their target cells is shown in Figure 20–3. The majority of steroid hormone that enters the cell is derived from the small nonbound fraction in the circulation, which can enter by passive diffusion. Upon entry into the cell, the steroid or its metabolic derivative (eg, DHT) binds directly to a predominantly cytoplasmic (androgen receptor) or nuclear (estrogen receptor) steroid receptor protein. The steroid-receptor complex undergoes an activation step involving a conformational change and shedding of heat shock (including HSP70, HSP90, and HSP40) and other chaperone proteins, which are necessary to maintain the receptor in a competent ligand-binding state (Aranda and Pascual, 2001). After dimerization, and nuclear transport in the case of some steroid receptors like the androgen receptor, the activated receptor dimer complex binds to specific DNA motifs called hormone-responsive elements (HREs) found in the promoters of hormone-regulated genes (Aranda and Pascual, 2001). The receptor-DNA complexes, in turn, associate dynamically with coactivators and basal transcriptional components to enhance the transcription of genes, whose messenger RNAs (mRNAs) are translated into proteins that elicit specific biological responses. It is likely that receptors that are not bound to their ligands, or those bound to antagonists (Fig. 20–4), form complexes with corepressors to inhibit the transcription of specific genes (Perissi and Rosenfeld, 2005).
Illustration of the role of coactivators and corepressor in regulation of steroid receptor action. In the presence of agonistic ligands (eg, estradiol for estrogen receptor [ER] and DHT for androgen receptor [AR]), the steroid receptor-DNA complexes associate dynamically with coactivators, which, in turn, recruit other proteins, including cointegrator complexes, that contact and stabilize the basal transcription unit resulting in enhanced transcription of target genes. In the presence of antagonistic ligands (eg, tamoxifen for ER and flutamide for AR), the receptors are in a different conformational state (steroid receptor ligand analog) and the receptor-DNA complexes dynamically associate with corepressors (Co-R) which destabilize basal transcription units and result in reduced transcription of target genes. HRE, hormone-responsive element.
All steroid receptors are members of a so-called superfamily of more than 150 proteins. All are ligand-responsive transcription factors that share similarities with respect to their structural homology and functional properties. As shown in Figure 20–5, each member of the steroid receptor family possesses a modular structure composed of the following:
An N-terminal region containing ligand-independent transcriptional activating functions (collectively referred to as activating function-1 or AF1);
A centrally located DNA binding domain of approximately 65 amino acids having 2 zinc fingers (see Chap. 8, Sec. 8.2.6);
A hinge region that contains signal elements for nuclear localization; and
A ligand-binding domain in the C-terminal region of the protein containing a ligand-dependent transcriptional activating function (called AF2).
The relative amino acid sequence homology within the functional domains of the principal members of the family of human (h) steroid hormone receptors. The relative amino acid sequence homology (represented by the percentages indicated in boxes) for hERβ is in reference to hERα, whereas all the other receptors are relative to hAR. The numbers correspond to the amino acid positions from the N-terminus (NH2), AF1 and AF2 refer to the transcriptional activation function 1 and 2 that reside in the N-terminal and ligand-binding domains, respectively. AR, Androgen receptor; ER, estrogen receptor; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PR, progestin receptor.
AF1 and AF2 can function independently or synergistically, depending on the gene promoter and/or the cell type (Aranda and Pascual, 2001). Between members of the family of steroid receptors, the N-terminal region has the highest degree of amino acid sequence variability, whereas the DNA-binding domain has the most shared homology.
220.127.116.11 Androgen Receptors
The androgen receptor (AR) is encoded on the X chromosome. As a single allele gene in males, it is susceptible to genetic defects whose phenotypes can range from minor undervirilization to a complete female phenotype known as testicular feminization. The normal, wild-type AR has a molecular weight of approximately 110 kDa, but truncated molecular variants have been observed in a variety of prostate cancers. These variants lack the ligand-binding domain of the AR but are still functional, even in the absence of androgen (Hu et al, 2009). In this chapter, AR refers only to the wild-type, 110-kDa form.
Relative to other steroid receptors, the AR has one of the largest N-terminal domains, which occupies more than half of the primary sequence of 919 amino acids (see Fig. 20–5). A unique feature of the AR relative to other members of this family is the occurrence of several stretches of the same amino acids (termed homopolymeric) in the N-terminal domain. These tracts include 17 to 29 repeating glutamine residues, starting approximately at amino acid 59, 9 proline residues at amino acid 372, and a 24-residue polyglycine stretch beginning at amino acid 449. These repeating tracts have been shown to modulate the folding and structural integrity of the N-terminal domain (Davies et al, 2008) and there is evidence of a reverse relationship between variations in their lengths and the transcription levels of mRNAs (Robins et al, 2008). Not surprisingly, these variations result in biological effects: for example, an abnormal extension of the polyglutamine tract to 40 or more residues is associated with neurodegenerative diseases such as X-linked spinal and bulbar muscular atrophy (La Spada et al, 1991). In contrast, a decreased size of these homopolymers may be linked with the development of prostate cancer (Robins et al, 2008), although there is no consensus on this relationship (Mir et al, 2002).
18.104.22.168 Estrogen Receptors
There are 2 estrogen receptor (ERs), ERα and ERβ, which, unlike AR isoforms, are encoded by separate genes (see Fig. 20–5). Several variant isoforms of each ER, generated by alternative RNA splicing, may also be expressed (Murphy et al, 1998). The centrally located DNA-binding domains for ERα and ERβ (see Fig. 20–5) are highly homologous (>95% identity). Their C-terminally located ligand-binding domains are approximately 60% identical. As with other steroid receptors, the ligand-binding domain contains a ligand-dependent dimerization function, and a ligand-dependent transactivation function, AF2. Similarly, a ligand-independent transactivation function, AF1, is present in the N-terminal domain. This latter domain is different in ERα and ERβ. In addition steroid receptors are subject to multiple posttranslational modifications such as phosphorylation, which can regulate receptor activity, transcriptional activity, DNA binding, protein turnover, and ligand sensitivity (Ward and Weigel, 2009).
20.2.5 Binding of Steroid Receptors to DNA
As transcription factors, steroid receptors modulate gene expression by first binding to specific DNA sequences in the promoter regions of hormone-regulated genes (see Fig. 20–4). The α-helix structure of the first zinc finger (see Chap. 8, Sec. 8.2.6) of the DNA-binding domain of a receptor is the primary discriminator for binding to different DNA sequence motifs of HREs (Aranda and Pascual, 2001). All members of the nuclear receptor family bind as dimers to pairs of a similar DNA sequence motif, AGNNCA (N = any nucleotide), which comprises the HRE found in the promoters of steroid regulated genes (Glass, 1994). Steroid receptors can bind to multiple HREs that are often hundreds of kilobase pairs distal to target gene promoters; furthermore steroid receptors and associated complexes bound at both distal and proximal HREs can interact, resulting in chromosome looping, to regulate target gene transcription (Fullwood et al, 2009). Nuclear receptors can be further subdivided on the basis of selection of the primary sequence of the core motif as either AGGTCA, for the ER and thyroid receptor subfamily, or AGAACA for the AR, glucocorticoid receptor (GR), or progestin receptor (PR). In general, 2 or more sets of interacting HREs in a gene promoter are required to elicit a steroid-mediated response (Klinge, 1999; Rennie et al, 1993).
The very high sequence homology of HREs has raised the question as to how steroid receptors govern hormone-specific responses. Although there is no definitive mechanism, the interaction with unique combinations of coregulators or other binding proteins is probably a major contributing factor to steroid-receptor specific gene regulation. Other parameters may also be important, such as the availability of receptor and ligand, the activity of proximal transcription factors, the cooperative binding of receptors to 2 or more DNA-binding sites, and altered DNA target motif recognition.
20.2.6 Interaction of Steroid Receptors with Coregulator Proteins
Important molecular mechanisms by which nuclear receptors regulate gene transcription involve not only direct interactions of steroid receptors with some of the basal transcription components, but also indirect interactions through recruitment of coregulator protein complexes to the promoters of target genes (Perissi and Rosenfeld, 2005; see Figs. 20–3 and 20–4). Coregulators fall into 2 main classes, coactivators and corepressors, which enhance or repress transcription, respectively (as illustrated in Fig. 20–4). There are many genes that encode coactivators, but the steroid receptor coactivator (SRC)/NCOA/p160 family are relatively specific for nuclear receptors and are the best studied (O'Malley and Kumar, 2009; Xu et al, 2009).
Although the auxiliary molecular components involved and the dynamics of their interactions remain to be established, a general pattern has emerged. The coactivators and corepressors are important for mediating both AF2 and AF1 activities of steroid receptors. Their mechanism of transcriptional activation is thought to involve 2 stages (Perissi and Rosenfeld, 2005). First, when recruited to the receptor by direct protein–protein binding, coregulators promote the local remodeling of chromatin structure through acetyltransferase or deacetylase activity and through their ability to recruit other proteins with chromatin remodeling activity (as outlined in Fig. 20–6). Second, the coactivators recruit and/or stabilize the basal transcription machinery by protein–protein interactions so as to enable efficient transcription of the target gene by RNA polymerase II (Perissi and Rosenfeld, 2005). The chromatin remodeling enables altered access of the promoter DNA to general transcription factors (Fig. 20–6).
Remodeling of chromatin and activation of transcription by steroid hormone receptors. Steroid hormone receptors bind to hormone responsive elements (HREs) in the promoters of target genes and recruit coactivators (eg, SRC-1) and cointegrators (eg, CBP/p300), which have chromatin remodeling activity. Some coregulators and cointegrators have histone acetyltransferase (HAT) activity, which results in dynamic nucleosomal histone acetylation and increased access of basal transcription complexes (that include RNA polymerase II) to the promoters of target genes.
It is uncertain as to which coactivators are necessary or sufficient for transcriptional activation. In MCF-7 breast cancer cells ERα and a number of coactivators associate rapidly with target promoters in a dynamic, cyclic fashion and the SRC/NCOA/p160 class of coactivators is sufficient for gene activation (Shang et al, 2000). It is likely that the relative availability of coregulators will vary in different tissues and may even be restricted to specific tissues. Also, although the occurrence of receptor-specific coregulators has not been confirmed, many coregulators bind preferentially to certain receptors (Leo and Chen, 2000). For example, a repressor of ER transcriptional activity, called repressor of estrogen receptor activity (REA) is active on ERα or ERβ, but not other steroid receptors (Montano et al, 1999). Its mechanism of action involves a competition with coactivators such as SRC-1 for binding to the ligand-binding domain of ER (Delage-Mourroux et al, 2000). Furthermore, a member of the SRC/NCOA family, SRC-3/NCOA3 or amplified in breast cancer 1 (AIB1) may have a role in breast cancer as it is amplified and overexpressed in some breast tumors and such overexpression has been correlated with tamoxifen resistance (Anzick et al, 1997; Xu et al, 2009).
20.2.7 Mechanisms for Transcriptional Regulation by Steroid Receptors
There are at least 3 different mechanisms by which steroid receptors regulate transcription of target genes:
Ligand-dependent and requiring direct binding of steroid receptors to HREs in promoter DNA (as described above and illustrated in Figs. 20–3 and 20–4);
Ligand-dependent but not requiring direct binding to DNA; and
Most hormone-regulated genes have ligand-dependent binding to HREs. Examples of target genes regulated in this fashion are prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA). However, in some target genes, ligand-activated ER regulates transcription, without contacting the DNA directly, through protein–protein interaction with other transcription factors that are in direct contact with DNA via their own specific response elements. For example, ERα can interact with Sp1 and AP-1 transcription factors and regulate transcription of some genes.
Ligand-independent activation of ER and AR can occur through crosstalk with a variety of growth factor networks. Growth factors such as epidermal growth factor (EGF) and/or insulin-like growth factor (IGF)-I bind to their respective tyrosine kinase receptors located in the plasma membrane of target cells and activate signal transduction pathways involving activation of other kinases (see Chap. 8, Sec. 8.2); these events can lead to phosphorylation of steroid receptors. One enzyme activated by growth factor signaling that can phosphorylate directly both ERs as well as the AR is mitogen activated protein kinase (MAPK) (Ueda et al, 2002). Similarly, interleukin-6 and protein kinase A can activate AR and ERα directly in the absence of the appropriate steroid ligand. Interleukin-6 may bind to and influence AR activity without inducing phosphorylation of the AR (Ueda et al, 2002). Growth factor/phosphorylation pathways can also influence steroid hormone receptor pathways via their ability to modulate coactivators by phosphorylation (Han et al, 2009). These alternative pathways for regulation of ER and AR activity could have a profound influence on the emergence of hormone independence in tumors that have not lost their hormone receptors (see Sec. 20.5.5).
20.2.8 Nontranscriptional Actions of Steroid Receptors
There is evidence that not all effects of steroid hormones are mediated via the regulation of genomic or transcriptional events (Kelly and Levin, 2001). Transcription-independent effects of estrogen manifest themselves as rapid responses in target cells of the order of seconds to a few minutes. For estrogen, examples of nongenomic effects include modulation of calcium ion flux, effects on membrane channels in the central nervous system and peripheral excitable cells, membrane-associated interactions with growth factor receptors, and interactions with survival/apoptosis pathways. There is evidence that membrane-associated ERs coupled to G proteins or nitric oxide-generating systems mediate some of these actions. At least 2 categories of membrane-based receptors may exist: one related to the classical intracellular ERα or ERβ (Kelly and Levin, 2001), and the other distinct from them (Nadal et al, 2000). Other steroid hormones such as progesterone and testosterone may also influence cells via related mechanisms (Kousteni et al, 2001).
The importance of nontranscriptional actions of estrogen in breast cancer in relation to estrogen-dependent signaling is not known. There are multiple levels of estrogen interaction with growth factor receptor kinases and the signal transduction pathways that they regulate. Therefore, nongenomic and genomic actions of estrogen are likely to be integrated with and complementary to each other. Unravelling this molecular complexity has important implications with respect to new therapeutic combinations and approaches (Arpino et al, 2008).
20.2.9 Quantification of Steroid Hormone Receptors
ER and PR are biomarkers in breast cancer. In particular, they help to predict the likelihood of response to endocrine therapy. In an unselected group of breast cancer patients with advanced disease, 30% to 40% will respond to endocrine therapy. However, patients whose tumors are both ER and PR negative have less than 10% chance of responding to endocrine therapy while in patients selected for the presence of both ER and PR in their primary breast tumor, the response rate to endocrine therapy is 70% to 80%.
Approximately 90% of unselected men with prostate cancer will respond to endocrine therapy: AR provides no prognostic or diagnostic value since almost all tumors usually possess a functioning AR, and almost all men respond initially to androgen withdrawal.
ER and PR are measured routinely in breast tumor biopsies by immunohistochemistry (IHC) using well characterized, specific monoclonal antibodies, as outlined in Figure 20–7A. IHC methods (Fitzgibbons et al, 2010; Hammond et al, 2010) can determine whether the detected receptor is within tumor cells, and can show receptor heterogeneity in tumor tissue. An example of ER heterogeneity within a breast tumor is shown in Figure 20–7B. Heterogeneity refers to the observation that both ER+ and ER– breast cancer cells can be present to varying degrees within any breast cancer biopsy sample, in addition to the presence of other types of cells (vascular cells, infiltrating cells of the immune system, normal stromal fibroblasts, normal breast adipocytes, and normal breast epithelial cells). Semiquantitative methods have been used to evaluate ER and PR by IHC, but positive results are generally reported when more than 1% of tumor nuclei stain positively (Hammond et al, 2010). There is a correlation between benefit and increasing ER level, which can be achieved by combining the level of staining intensity with the percentage of tumor cells positively stained to give H-scores or Allred scores (Hammond et al, 2010). Results can vary amongst laboratories, especially in the low to middle range of the receptor spectrum, and guidelines for standard operating procedures for tissue collection, assay validation, quality control, and interpretation have been published (Fitzgibbons et al, 2010; Hammond et al, 2010).
Immunohistochemical–Avidin-biotin complex method. A) Determination of hormone receptors in tumor tissue. Thin sections of tumor are cut from formalin-fixed, paraffin-embedded biopsy specimens. The section is next exposed to a monoclonal antibody specific for the steroid receptor (SR) being assessed. The section is then exposed to a second biotinylated antibody specific for the first antibody. Finally, avidin-peroxidase complex is added, followed by a chromogen, and color appears where the SR is located. B) Immunohistograms illustrating heterogeneity of human breast cancer biopsy samples. Brown staining represents ERα positivity. T, Invasive breast cancer; S, stromal and connective tissue elements; L, lymphocytes. (i) Homogenous expression of ERα within an invasive breast cancer, with negative adjacent vessels and stroma. (ii) Moderate heterogeneity of expression of ERα within an invasive breast cancer, with strong expression within solid nests of tumor cells in the upper field and weak or negative expression within less-cohesive clusters of tumor cells in the lower field. Stromal and lymphocytic elements are negative for ERα. (iii) Marked heterogeneity of expression of ERα within an invasive tumor metastatic to an axillary lymph node. In the upper part of the section one metastatic component is homogenously ERα –ve and in the lower part of the section the other component is moderate to highly ERα +ve. These 2 different elements are separated in this field of view by a band of fibrous stroma and infiltrating lymphocytes.
ER and PR can also be assayed by measuring their mRNA. This is currently being assessed as part of a 21-gene expression analysis, known as Oncotype DX, using reverse-transcription and quantitative polymerase chain reaction technology (see Chap. 2, Sec. 2.2.5). This evaluation is only carried out by the company that developed the assay, which generates a recurrence score (RS) that helps predict the benefit of adding chemotherapy to hormonal therapy in women with ER+ breast cancer (Paik et al, 2006; Albain et al, 2010). The clinical utility of the Oncotype DX assay and other multigene assays is still being evaluated in prospective clinical trials (TAILORx [Trial Assessing Individualized Options for Treatment for Breast Cancer]; MINDACT [Microarray in Node-Negative and 1-3 Node Positive Disease May Avoid Chemotherapy Trial]) (Cardoso et al, 2008).