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The stage of a cancer is one of the most important factors in determining prognosis and treatment options. The most common system used to describe the stages of breast cancer is the American Joint Committee on Cancer (AJCC) TNM classification system. It is used by doctors to determine the stage of different types of cancer based on certain standard criteria (Greene et al., 2002). The TNM system is based on 1) the extent of the tumor (T), 2) the extent of spread to the lymph nodes (N) and 3) the presence of metastasis (M). A number is added to each letter to indicate the size or extent of the tumor and the extent of spread (National Cancer Institute, 2007e).
The stage of a breast cancer is determined either by the results of physical examination, biopsy and imaging tests, which collective is called the clinical stage, or by the results of the clinical stage tests together with the results of surgery, which is called theÂ pathologic stage. The staging described in Figure 2.2 is the pathologic stage, which includes the findings after surgery, when the pathologist has looked at the breast mass and nearby lymph nodes. Pathologic staging is likely to be more accurate than clinical staging, as it allows the doctor to get a firsthand impression of the extent of the cancer (http://www.cancer.org/Cancer/BreastCancer/DetailedGuide/breast-cancer-staging).
Table 1.1 describes the stages of breast cancer and the definition of each stage
Table 1.1 Stages of breast cancer
Cancer cells remain inside the breast duct, which has not spread into normal adjacent breast tissue.
Cancer is 2 centimeters or less and has not spread to any lymph nodes.
The tumor is 2 centimeters or smaller and has spread to the axillary lymph nodes OR
the tumor is between 2 - 5 centimeters and has not spread to any lymph nodes.
The tumor is between 2 -5 centimeters and Stage has spread to the axillary lymph nodes OR
the tumor is larger than 5 centimeters but has not spread to lymph nodes and does not grow into chest wall.
No tumor is found in the breast. Cancer is found in axillary lymph nodes that are sticking together or to other structures, or cancer Stage may be found in lymph nodes near the breastbone OR
the tumor is any size. Cancer has spread to the axillary lymph nodes, which are sticking together or to other structures, or
cancer may be found in lymph nodes near the breastbone.
Tumour has grown and has spread to the chest wall and/or skin of the breast AND
may have spread to axillary lymph nodes that are clumped together or sticking to other structures, or cancer may have spread to lymph nodes near the breastbone.
Inflammatory breast cancer is considered at least stage IIIB.
There may either be no sign of cancer in the breast or a tumor may be any size and may have spread to the chest wall and/or the skin of the breast AND
the cancer has spread to lymph nodes either above or below the collarbone AND the cancer may have spread to axillary lymph nodes or to lymph nodes near the breastbone.
The cancer has spread to other parts of the body (metastasis).
Adopted from (Kumar et al., 2005; Kwanyoung, 2009; Greene et al., 2002).
2.2.6 Diagnosing Breast Cancer
The usual methods that are used to detect breast cancer include the use of self examination of breast, mammography, positron emission tomography (PET) scans, magnetic resonance imaging (MRI), ultrasound, and breast biopsy. Nevertheless, there are not one of these tests by themselves can decide with certainty the existence of cancer.
Mammography is a specific type of non-invasive radiograph (X-ray) system to examine breast tissue .It is used as a diagnostic and a screening instrument if cancer is existent. Mammography of the breast tissue produces a photographic or digital image of the breast tissue and has been considered the gold standard method in diagnosis and early detection of breast cancer (Nass et al., 2001). To have an early detection of breast cancer, mammography is used in the general population. This is regarded as a basis which shows stable decrease in tumor size and lower stage at diagnosis. It has been also considered as an increase in ductal carcinoma in situ in all series, and the maturation of survival data from multiple trials (Lawrence, 2001). Most women could identify breast masses by themselves during the breast self-examination. Yet, most masses identified are not cancerous. It has been believed that tumors of breast can be found and reported by women who palpate their breasts at regular basis and that the likelihood of treatment will thus be improved (Hill et al., 1988). Breast self-exams alone do not reduce the number of fatality from breast cancer and can overlook tumors. Consequently, more than one method to screen for breast cancer is always essential.
It has been reported that a combined approach to breast cancer screening, which includes clinical breast exams, mammography and magnetic resonance imaging (MRI) for certain high risk women and breast self-exams, increases their chances of getting breast cancer at an curable stage (Harris and Kissenger, 2002). Adding biomarker with high specificity could reinforce the chances of precisely diagnosing breast disease and detecting the disease at an early stage.
The ultrasound is a procedure that produces high-frequency sound waves through the breast tissue to transfer the image onto a screen. It is used to complement other tests such as mammography. In conditions where a mammogram displays an abnormality that requires further definition, an ultrasound is often beneficial to assess whether a density that shows on a mammogram is a fluid-filled cyst, a solid mass, or difference of normal breast tissue. A cyst is generally benign while a solid mass may be either benign or malignant. Yet, ultrasound is not enough for routine screening because it cannot explore the whole breast at one time. It can only visualize small areas exactly. In addition, it is not as detailed as a mammogram. It should be considered that 1%-2% of breast cancers are not identified by an ultrasound or mammography (Berg et al., 2008). Thus a biopsy may be necessary when there are no clear results from an ultrasound or mammogram.
The Positron Emission Tomography Scans (PET) is considered one of the newest diagnostic methods of the breast cancer which is still in the experimental stage. To help radiologists detect cancer, it is necessary to inject radioactive material into the potentially malignant cells. Yet, there are pitfalls to this method. First, PET scans have a limited capability to detect small tumors (Van der Hoeven et al., 2002). Second, PET scans exists in only very few centers and finally, they are sophisticated, and expensive test that requires exceptional knowledge.
Another alternative method of diagnostic breast cancer is the Magnetic Resonance Imaging (MRI) which is used for radiation. This method used magnetic fields to create images of the body. It has been reported that Breast MRI has been shown to be capable of detecting early breast cancer (Smith and Andreopoulou, 2004). The Breast MRI method depends on assessing how blood flows into and out of different types of tissue in the breast through injecting a gadolinium dye into a vein. (Caravan et al., 1999).
The last method for breast cancer diagnostic is the biopsy whereby cells or tissues are removed for examination. The biopsy is used to determine the existence and confirm the progression or the removal of the whole area of the diseased tissue. It is conducted to detect a suspicious abnormality through mammography scan. Having the tissue removed, it can be examined by a pathologist who determines the diagnosis. There are various methods that could be performed for breast biopsy. This depends on the features of the abnormality. The methods involve fine needle aspiration (FNA), core, incisional, open surgical, vacuum assisted, and minimal invasive biopsy. However, stereotactic breast biopsy is considerably used as a substitute to surgical biopsy, there are concerns regarding its usefulness in the assessment of calcifications highly suggestive of malignancy (Liberman et al., 2001). One of the main disadvantages of breast biopsy, is that even if the biopsy results are non-cancerous, that is benign, patients would feel as though they have undergone an invasive procedure unnecessarily. There was a shared effort by radiologists and surgeons to be more selective when deciding candidates for breast biopsy, there would still be relatively high negative biopsy rates. This can be overcome through new technology used in place of breast imaging evaluation (Lawrence, 2001).
2.3 Breast Cancer Biomarkers
Apart from the various diagnostic methods that could be used to detect breast cancer as described above, another method that could be used is biomarker. Biomarkers are genes, protein, peptides or metabolite found in a biological system. It signifies the physiological and pathological changes during the presence of disease. The ability of biomarker to be used as an indicator helps to monitor disease conditions and assists in its diagnosis and prognosis. The appropriate biomarkers can be used to complement and assist tumor detection and diagnosis, predict the outcomes of the disease, define the risks and identify the early stages of tumor development, verify stratification of patients for treatment and help in surveillance for disease recurrence (Victor & Levenson, 2007).
In this regard, the Food and Drug Administration (FDA) of the United States of America has approved only a few new diagnostic biomarkers to be used in cancer related research and diagnoses. They are:
Carcinoembryonic antigenÂ (CEA) for malignant pleural effusion (Li et al., 2003) and peritoneal cancer insemination (Yamamto et al.2004)
Human Epidermal Growth Factor ReceptorÂ 2 (Her-2/neu )for stage IV breast cancer (Cook et al. 2001), Bladder tumor antigen for urothelial cell carcinoma (Mian et al., 2000), Thyro-globulin thyroid cancer metastasis(Lima et al.2002),
Alpha-fetoprotein for Hepatocellular carcinoma (De Masi et al., 2005),
Prostate-specific antigen (PSA) for prostate cancer (Cann et al., 1995),
cancer antigen (CA 125) for Non-small cell lung cancer (Dabrowska et al.2004),
CA19.9 for pancreatic cancer (Yamagushi et al., 2004),
CA 15.3 for breast cancer (Ciambellotti et al., 1993),
Leptin, prolactin, osteopontin and Insulin-like growth factor 2Â (IGF-II) for ovarian cancer(Mor et al., 2005),
CD98,Fascin,sPIgR4, and 14-3-3 eta for lung Cancer(Xiao et al., 2005),
Troponin I for myocardial infarction (Eggers et al., 2002),
B-type nariuretic peptide for Congestive heart failure (Dao et al., 2001). (Malu Polanski and Anderson, 2009).
The American Cancer Society reported that breast cancer in the United States is the most widely spread type of cancer diagnosed in women (American Cancer Society, 2010). While most of patients are diagnosed with early stage disease; yet, many will develop systemic recurrence later on. Therefore, such a case underpins the value of serial monitoring for recurrence of breast cancer using circulating tumor markers. However, attempts to find out such serum biomarkers in breast cancer have considerably been unsuccessful except for the development of immunoassays for CA15-3, CA27-29 and carcinoembryonic antigen (CEA) for monitoring patients with early and advanced breast cancer (Hou et al., 1999; Einarsson et al., 2000). The test of CEA blood measures the level of the antigen CEA by a sandwich enzyme linked immunoassay. On the other hand, the CA15-3 and CA27-29 tests measure the serum level of a mucin like membrane, glycoprotein (MUC-1) that is shed from tumor cells into the bloodstream. The CA 15-3 epitope is realized by two monoclonal antibodies in a double-determinant or sandwich immunoassay. The CA27-29 is a one epitope antibody test that is produced against MUC-1 protein.
According to Hayes, (1986); and Wojtacki et al., (2001), it is widely acknowledged that 75% to 90% of patients with metastatic breast cancer will have elevated MUC-1 levels. In this regard, several studies have shown that a rising CA15-3 or CA27-29 level can detect recurrence after primary treatment. Thus, tests measuring MUC-1 have been used in the management of patients with breast cancer. For example, Hou et al (1999) revealed that in patients with metastatic breast cancer, the sensitivity and specificity was 85.7% for CA27-29, 82.8% for CA15-3 and 62.8% for CEA, respectively. In addition patients had considerably higher levels of CA27-29 than CEA, but their CA27-29 levels were similar to CA15-3 which implies that CA27-29 is more sensitive and specific than CEA, but is similar to CA15-3 for metastatic breast cancer detection and monitoring (Hou et al., 1999).
According to Bensouda et al.'s study (2009), patients who have hormone Receptor (HR) sensitive and Human Epidermal Growth Factor Receptor 2 (HER2) negative tumors were demonstrated to be more likely to have elevated CA15-3 level at the time of diagnosis of metastatic disease than patients with other tumor types (Bensouda et al., 2009). In another study, Safi et al (1989) point out that CA15-3 serum levels preoperatively in N = 1342 patients with benign breast conditions and various malignancies. The findings indicated that CA15-3 levels were found to be over 50 U/ml in 0%, 2%, 13%, and 73% of the patients with stages 1, 2, 3, and 4 breast cancers respectively. Other study has revealed that in patients with liver metastases, CA15-3 levels were increased and that increased CA15-3 concentration usually preceded the clinical diagnosis of the relapse with the median lead time of 9 months (range: 1-40) in 72.4% of patients with distant metastases due to breast carcinoma (Wojtacki et al., 2001). However, in early stage, the low detection rate of CA15-3 breast cancer has prevented its routine use for screening breast cancer recurrence in spite of the fact that CA15-3 is used to monitor the effectiveness of treatments for metastatic breast cancer in addition to imaging studies and clinical symptoms.
Other recently developed serum-based tumor markers used in breast cancer detection and diagnosis which are based on enzyme immunoassays include the plasminogen activator (PA) system which is made up of the 2 serine proteases, urokinase PA (uPA) and tissue PA (tPA), the 2 serpin inhibitors, PAI-1 and PAI-2 and the uPA receptor (uPAR; CD87). High levels of uPA, PAI-1, uPA-PAI-1 complex and uPAR in breast cancer tissue are associated with poor prognosis while high levels of tPA or PAI-2 are associated with good prognosis (Meijer-van et al., 2004).
Tumor markers that are used in the detection and diagnosis of breast cancer are varied in number and type. They include mucins e.g. CA15.3 (Safi et al, 1991;Clinton et al, 2003); CA 27-29 (Frenette et al, 1994); oncofoetal proteins (e.g. CEA) (Esteban et al, 1994; Sundblad et al, 1996); oncoproteins e.g. Her-2 (Muller et al, 2006; Kong et al, 2006; Hudelist et al, 2006 ;Imoto et al, 2007); c-myc (Breuer et al, 1994); p53 (Hassapoglidou et al, 1993; Balogh et al, 2006); cytokeratins e.g. TPA (Nicolini et al, 2006; Sliwowska et al 2006) and estrogen receptor (ESR) (Robertson et al, 1991 and 1999; Rubach et al, 1997). More recent tumor markers described in the literature include Mammaglobin (Watson et al, 1996), survivin (Goksel et al, 2007; Yagihashi et al, 2005), livin (Yagihashi et al, 2005), NYESO- 1 (Bandic et al, 2006), Annexin XI-A (Fernández-Madrid et al, 2006), Endostatin (Balasubramanian et al, 2007), Hsp90 (Pick et al, 2007), p62 (Rolland et al, 2007) and koc (Zhang et al, 2003).
However, currently, there are very few serum markers that are used clinically for breast cancer. Some studies have identified as possible breast cancer markers the proteins CA 15.3 (Duffy, 2006; Cheung et al., 2000), BR 27.29(CA 27.29), tissue polypeptide antigen (TPA), tissue polypeptide specific antigen (TPS), shed HER-2 (Cheung et al., 2000), and BC1, BC2, and BC3 (Li J, et al., 2002; Mathelin C, et al., 2006). However, other studies found a lack of sufficient diagnostic ability in serum proteins, including CA 15.3 (Mathelin C, et al.,2006; Skates SJ, et al.2007), CA 125 (Skates SJ, et al.2007), CA 19.9 Skates SJ, et al.2007), CA 125 Skates SJ, et al.2007), BR 27.29 (Duffy ,2006; Molina R, et al.,2005), and carcinoembryonic antigen (CEA) (Duffy ,2006).
When breast cancer is detected in its early stages, it can be treated (Levenson, 2007). However, there are currently no FDA approved serum tests for early detection of the disease. To add to the problem, the early symptoms of breast cancer are sometimes absent or not recognized. It is frequently detected in an advanced stage of progression and hence untreatable by the time the cancer is finally diagnosed (Kirmiz et al., 2007).
In breast cancer, the presence of increasing concentrations of highly glycosylated proteins (mucins) along with other changes in glycosylation are associated with increasing tumor burden and poor prognosis (Hollingsworth and Swanson, 2004). Glycosylation of proteins is known to change in breast and other types of cancer (Hakomori 2001; Hollingsworth and Swanson, 2004). Alterations in glycosylation influence growth, differentiation, transformation, adhesion, metastasis and immune surveillance of the tumor (Choudhury et al., 2004). O-linked glycosylation of the mammary gland is altered during malignancy due to the changes in mucin glycosylation (Burchell et al., 2001).
Mucin 1(MUC1), CEA (carcinoembryonic antigen) and mammaglobin are implicated in breast cancer as Serum biomarkers (Bernstein et al., 2005; Duffy, 2006). The immunoassay tests for MUC1 (CA27.29 or CA15-3) and carcinoembryonic antigen (CEA) are only serum tests approved to be used in breast cancer. For example, The European Group of Tumor Markers identified the MUC-1 mucin glycoproteins CA 15.3 and BR 27.29 as the best serum markers for breast cancer. However due to their low sensitivity, these proteins could not be recommended for diagnosis (Molina R, et al., 2005). Similarly, as Kirmiz et al., (2007); Dâ€ŸArcy et al., (2006) point out, current available tumor markers lack the specificity and sensitivity to enable them to be used in early detection of breast cancer. According to Duffy (2006),
These serum markers are also not recommended by the Association of Clinical Oncologists and are only approved for use to monitor treatment of patients with breast cancer. Therefore, it is imperative that a reliable biomarker is available to be used to rule out breast cancer in the early stage.
MUC1 is understood as the polymorphic epithelial mucin (PEM). In addition, it is the target of a test for pancreatic, hepatic and colon cancers (CA19-9). The polymorphic nature or "heterogeneity" of PEM is essentially because the high amounts of O-linked glycosylation of the tandem repeat elements present in the extracellular carboxyl end of the molecule (Taylor-Papadimitriou et al., 1999). The CA27.29 and CA19-9 tests are detected different MUC1 antigenic epitopes that correspond to the different types of cancer by using antibodies. Furthermore, present on many of these proteins and detectable by antibodies are N-linked oligosaccharides (glycans) that is a different variety of glycosylation that is also implicated in cancer (Kobata and Amano. 2005).
2.4 Proteomics of Breast Cancer
Proteomics is the study of structure and function of all proteins encoded by genome in a cell or tissue (Dwek and Rawling, 2002,Karp and Lilley, 2007). Or as Carpenter and Melath (2003), define it, proteomics is the scientific study of biological diseases using qualitative and quantitative comparison of proteomes under two or several different conditions, for example, normal versus diseased.
The wealth of information generated by genomics in breast cancer can be complemented and further enlarged by proteomics for several reasons. Its major advantage if compared with genomics is that proteins are more reflective of the existing condition of the cell's microenvironment, where the levels of gene activity do not exactly correlate to the corresponding protein expression levels. In other words, mRNA levels do not necessarily correlate with corresponding protein abundance (Kennedy, 2001; Nishizuka et al., 2003; DaoHai et al., 2006 and Larong and Dark, 2007). This is the case, because, additional complexity results from protein post-translational modifications, including phosphorylations, acetylations, and glycosylations, or protein cleavages (Tyers and Mann, 2003).
2.4.1 Post-translation modifications and cancer
Most proteins suffer some form of post-translational modification (PTM) during their biological life. It can be noted that some of the important roles of PTM's can be found in cell signaling, generation of active forms of proteins, coding proteins for transport to specific compartments, and in making polypeptides for degradation (Collins and Choudhary, 2007). Thus, on account of all these important functions, the formation, fate, and role of post-translationally modified proteins in cellular regulation are major issues in proteomics.
Moreover, there are a number of ways to conduct a global study of all PTMs of a particular class. Peptides or proteins that bearing a particular modification often have a unique mass spectral fragmentation signature that is relatively easy to identify, for example, as in the case with phosphorylation (Temporini et al., 2008). The disadvantage of this approach is the need to perform a MS/MS analysis of every peptide in the sample so as to identify a small number that are phosphorylated, unless the phosphorylated peptides have been selected and improved.
A lot more troublesome type of post-translational modification to identify is one that varies in structure as is the case with protein oxidation or glycosylation (Owen et al., 2009). Indeed not only can proteins be oxidatively modified in dozens of different ways (Stadtman, (1997), but also that lectin arrays indicate that glycosylation is even more complex (Patwa et al., 2009). Usually, both the type and number of monosaccharide residues appearing in the glycan appended to a particular site on a polypeptide are variable. For example, usually there are 10-50 glycoforms at a single site on a protein (Yang, 2004), because glycan structure is determined by the sequential addition and trimming of sugars from the glycan by a series of glycosyltransferases and glycosidases (Taylor and Drickamer, 2003). In other words, there are substantial variations in the structure of a glycan. In contrast, polypeptide structure is determined by a single mRNA template.
Apart from glycosylation, the other post-translational modifications (PTMs) include phosphorylation, methylation, acylation, oxidation and ubiquitinylation. During cancer progression, many PTMs contribute to abnormal cellular proliferation, adhesion characteristics and morphology (Krueger and Srivastava, 2006; Golks and Guerini, 2008).
In this regard, of particular interest is that recent studies in breast cancer suggest that PTM profiles can be used as "biochemical footprints" for tracking and verifying the function and activity of key cellular signaling pathways (Hanash et al., 2008; Spickett et al., 2006). One important implication of such a finding is that PTMs may be useful biomarkers for the detection of early breast cancer.
2.4.2 Phosphoproteomics of Breast Cancer
Protein phosphorylation is a reversible covalent modification that affects almost 30% of the proteins expressed in mammalian cells. The importance of this modification is underscored by the fact that there are more than 500 genes in the human genome encode protein kinases and as many as 100 genes encode protein phosphatases (Janke et al., 2008).
Phosphorylation is one of the commonest post-translational modifications involved in regulating biological processes in a cell. The dysregulation of kinase signaling pathways is usually associated with various cancers (Hanahan and Weinberg, 2000). For example, aberrations in kinases have been reported in several cancers including gastrointestinal stromal tumors (Corless et al., 2004), lung cancer (Sharma et al., 2007), haematologic malignancies (Ferrajoli et al., 2006), breast cancer (Hynes and MacDonald, 2009), pancreatic cancer (Harsha et al., 2008) and prostate cancer (Lee et al., 2008).
For example, these aberrations may be caused by over expression of kinases, mutations or defects in negative regulatory mechanisms. Activated kinases can be specifically targeted using small molecule inhibitors. Some examples of such targeted therapeutic approach employing small molecule kinase inhibitors have been reported in the treatment of various cancers including chronic myelogenous leukemia (CML) (Druker et al., 1996; Golas et al., 2003), gastrointestinal stromal tumors (Braconi et al., 2008), small cell lung cancer (Krystal et al., 2000), breast cancer (Xia et al., 2002; Rabindran et al., 2004;), non-small cell lung cancer (Lynch et al., 2004) and melanomas (Karasarides et al., 2004).
A number of proteomic approaches have been developed over the years to identify aberrantly activated kinases and their downstream substrates. Currently, it is common to us phosphorylation as a surrogate for monitoring kinase activity in cells. Previously, kinases and their activities were generally studied on an individual basis using biochemical approaches. Recent technological advances have led to the development of several high throughput strategies to study the phosphoproteome. Examples of high-throughput technologies for monitoring phosphorylation events include array-based technologies such as peptide arrays (Houseman et al., 2002; Diks et al., 2004; Amanchy et al., 2008; Versele et al., 2009), reverse-phase protein arrays (Gulmann et al., 2009), antibody arrays (Gembitsky et al., 2004; Zhong et al., 2008) and mass spectrometry (Loyet et al., 2005; Rikova et al., 2007;Chen and Yates, 2007;Molina et al., 2007; Harsha et al., 2008;Choudhary et al., 2009;Pan et al., 2009). The advantage of quantitative phosphoproteomic profiling is that is allows researchers to investigate aberrantly activated signaling pathways and therapeutic targets in cancers.
Phosphorylation is widely recognized as a key regulator of enzyme activity as evident in the findings extensive research in protein phosphorylation (Lange et al., 2008; Glover and Lee, 2004). The abnormal phosphorylation of defined signal transduction pathways can alter the growth properties of breast tumors. The analysis of protein phosphorylation profiles using sequence-specific antibodies against phosphorylation sites, allow us to determine the activation status of signaling pathways, which can provide valuable prognostic insights (Vazquez-Martin et al., 2008; Ouyang et al., 2001). For example, Atsriku et al (2008) undertook a systematic mapping of PTMs in the human estrogen receptor alpha (ER-Î±) in the MCF7 breast cancer cell line. They used HPLC-ESI and MALDI-MS techniques to identify the phosphorylation sites on the estrogen receptors in these cells. The use of both HPLC-ESI and MALDI gave higher sequence coverage than either approach alone. Their experiment identified nine phosphorylated serine residues, of which three were previously unreported.
As an alternative to current immunochemical or proteomic methods for finding biomarkers of breast cancer in patient serum, global profiling methods for glycans cleaved from their protein core are being developed (Cancilla and Lebrilla ,1998). The resulting free glycan species can be directly analyzed by mass spectrometry, thereby creating a profile of glycans, some of which are biomarkers for breast cancer.