Deciphering the molecular signaling pathways in breast cancer pathogenesis and their role in diagnostic and treatment modalities

Accepted Manuscript Deciphering the molecular signaling pathways in breast cancer pathogenesis and their role in diagnostic and treatment modalities

A.G Thivyah Prabha, Durairaj Sekar PII: DOI: Reference:

S2452-0144(17)30004-3 doi: 10.1016/j.genrep.2017.01.003 GENREP 114

To appear in:

Gene Reports

Received date: Revised date: Accepted date:

9 October 2016 20 December 2016 20 January 2017

Please cite this article as: A.G Thivyah Prabha, Durairaj Sekar , Deciphering the molecular signaling pathways in breast cancer pathogenesis and their role in diagnostic and treatment modalities. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Genrep(2017), doi: 10.1016/j.genrep.2017.01.003

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Deciphering the Molecular Signalling pathways in Breast cancer pathogenesis and their role in diagnostic and treatment modalities. Thivyah Prabha A.G 1 Durairaj Sekar 2

Department of Biochemistry,Narayana Medical College and Hospital, Nellore,

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2

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India.

Department of Biotechnology, School of chemical and Biological Sciences, REVA

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University, Bangalore - 560064.

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Dr. Thivyaprabha A.G

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Corresponding Author*

Department of Biochemistry

Nellore

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Narayana Medical College and Hospital

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India. Email: [email protected] Phone: 9686653148.

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ABSTARCT: Breast cancer is more common among urban females. It has been estimated that in India there will be 1,55, 000 new cases of breast cancer and 76,000 deaths due to breast cancer in 2015. Many factors play a key role in the progression of breast cancer but there is no evidence that proves or cure its progressions. Therefore, It is very important to take necessary early steps to

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screen , diagnose and treat the breast cancer as early as possible to save millions of lives. The

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present review high lights the significance of so many factors responsible for the progression of

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this disease, which includes pathological causes, roles of various molecular and biochemical markers, genetic mutations, role of micro RNA, gene expression analysis, cancer stem cells, apoptotic markers, prognosis, treatment and also emerging treatment modalities along with the

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existing ones like vaccines.

KEY WORDS: Breast cancer, Tumour markers, microRNA, Metastasis, Molecular

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Markers,Genes

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1. INTRODUCTION

Breast cancer is characterised by the uncontrolled growth of abnormal cells in the milk producing glands of the breast or in the passages (ducts) that deliver milk to the nipples. The type of breast cancer is important in determining the most effective treatment approach. The most common way to classify breast tumours is according to the status of three specific cell surface

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receptors. Every year more than one million women are diagnosed with breast cancer worldwide

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and over half of whom will die from the disease.[1] Breast cancer is the most common cancer

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and the leading cause of cancer death for women.[2] The average 5 year survival rate for women with late stage or advanced breast cancer remains low. Treatment options for breast cancer vary

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depending on the stage at which the cancer is diagnosed. This article provides an overview of breast cancer, including prevalence, risk factors, symptoms, pathogenesis, diagnosis, tumour

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markers and treatment options.

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1.1 Breast cancer prevalance

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Breast cancer is more common among urban females. It has been estimated that in India there will be 1,55, 000 new cases of breast cancer and 76,000 deaths due to breast cancer in 2015. [3]

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It is important to detect the breast cancer early to save millions of lives. Stigma, limited awareness, knowledge and lack of population wide screening program have led to late detection

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of most breast cancers.[4] The breast cancer presentation in the Indian population is widely reported to be around 10 years younger compared to the developed world.[5] More treatment

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choices are available for the early detected breast cancers and also there are greater chances of long-term survival.[6] At early stage detection ,breast cancer is curable, with a 100% survival rate for stage 0 and 1.[7] Studies have suggested that in the Indian scenario, the shift to routine use of mammography as a screening tool may not be justified.[8,9] Hence there arises a need to seek for a better marker for breast cancer diagnosis at early stages of the disease. 2. BREAST CANCER RISK FACTORS

The second most common cause of cancer death in women, and the main cause of death in women ages 40 to 59 as breast cancer is most common in females.[10] If breast cancer was

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diagnosed first during pregnancy, mortality rate from breast cancer has been significantly greater compared with those who had never been pregnant.[11] The lifetime probability of developing breast cancer is one in six overall [12] factors including prenatal conditions, diet, physical activity, estrogen exposure, body mass index, depression and quality of life have been mentioned as breast cancer risk factors.The main risk factor is a positive family history . High amounts of alcohol, fat, caffeine and red meat containing diet are all the positive risk factors for bearing

reducing

breast cancer.[13,14]

Long duration exposure to higher concentrations of

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in

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breast cancer. Diet with phytoestrogens and high amounts of calcium/vitamin D can be effective

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endogenous estrogen(menarche, pregnancy, and menopause) increases the risk of breast cancer. Testosterone level has also showed some parallelism with higher rate of breast cancer. Younger

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age of menarche and older age of first full-term pregnancy are associated with a higher risk of breast cancer. There is an increased risk of breast cancer in oral contraceptive users.

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Postmenopausal use of hormone therapy for long time is associated with increased risk of breast cancer. Short-term Hormone therapy appears not to increase the risk significantly. But it may

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make mammographic detection more difficult. Environmental toxic agents such as Organochlorines include polychlorinated biphenyls (PCB's), dioxins, and organo chlorine

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pesticides such as DDT are weak estrogens with high lipophilic properties and as a result, can store in adipose tissues. Exposure to these chemicals will increase the risk of breast cancer. [15]

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Age and gender are among the strongest risk factors for breast cancer. Breast cancer occurs 100 times more frequently in women than in men. Incidence rates increase with age until about the

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age of 45 to 50.

Breast cancer prevalence varies with the ethnic difference. Breast cancer is more common among

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whites. Women with higher educational, occupational and economic level are at greater risk because of their reproductive pattern including age of parity and age of first birth. Ethnic differences in estrogen and progesterone receptor subtypes have been also determined as important factors that affect the probability of breast cancer. The various status of estrogen receptor (ER) / progesterone receptor (PR) including ER-/PR-, ER+/PR+, ER-/PR+ and ER+/PR- have been reported . ER/PR status varied significantly across racial/ethnic groups even within the same tumor stage. High prevalence of hormone receptornegative tumors in African-American women may contribute to their high breast cancer mortality.[16] . Table.1 Represents the risk factor for breast cancer progressions.

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3. BREAST CANCER GENETICS Genome instability, whether inherited or not, results in a greater potential to develop genetic changes such as gene loss, gene amplification, point mutations and chromosomal translocations.[18,19] Inherited cancer syndromes often involve this phenotype, for example, the breast-cancer susceptibility gene 1 and 2 (BRCA-1 and BRCA-2) breast/ovarian cancer risk

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genes are both involved in DNA repair.[20,21] Markers have been classified into 2 groups

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depending on the role in the development of cancer, oncogenes and tumor suppressor genes.

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[22,23] An oncogene results from a gain of function mutation of a proto-oncogene that generates a tumorigenic product, whereas mutation of a tumor suppressor causes a loss of function in the

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ability to restrain cell growth. Many of the genes classified as oncogenes falls into categories of abnormally activated growth factors, growth factor receptors, intracellular signaling molecules transcription factors.

Tumor suppressor genes mostly related and known to

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and nuclear

influence the cell cycle machinery are pRb and p53[23] c-Jun is a protein that in humans is

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encoded by the JUN gene. c-Jun in combination with c-Fos, forms the AP-1 early response transcription factor c-jun overexpression is proposed to lead to an estrogen-independent

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phenotype in breast cancer cells. The observed phenotype for MCF-7 cells with c-jun overexpression is similar to that observed clinically in advanced breast cancer. Moreover, there

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has been much attention focused on oncogenic components of the cell signaling system, such as the HER-2/Neu cascade.[24,25]

shows genetic factors

involved in the breast

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cancer.[24,25].

Figure.1

3.1 ROLE OF BRCA1& BRCA2 IN BREAST CANCER

BRCA1 and BRCA2 are a human gene and its protein product, respectively. The official symbol (BRCA1, italic for the gene, nonitalic for the protein) and the official name (breast cancer 1, early onset) are maintained by the HGNC.[26] Orthologs, styled Brca1 and Brca1, are common in other mammal species.[27]BRCA1 is a human tumor suppressor gene (to be specific, a caretaker gene)[28,29], found in all humans; its protein, also called by the synonym breast cancer type 1 susceptibility protein, is responsible for repairing DNA.[30] BRCA1

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and BRCA2 are normally expressed in the cells of breast and other tissue, where they help repair damaged DNA or destroy cells if DNA cannot be repaired. They are involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double-strand breaks.[31,32] The BRCA1 protein associates with RNA polymerase II, and through the Cterminal domain, also interacts with histone deacetylase complexes. This protein plays a role in transcription, DNA

repair of

double-strand

breaks[31] ubiquitination, transcriptional

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regulation as well as other functions.[33] High rates of mutation occur in exons 11–13 called

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serine cluster domain (SCD). Reported phosphorylation sites of BRCA1 are concentrated in the

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SCD where they are phosphorylated by ATM/ATR kinases both in vitro and in vivo. ATM/ATR are kinases activated by DNA damage. Mutation of serine residues may affect localization of

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BRCA1 to sites of DNA damage and DNA damage response function. [34] In BRCA mutation, damaged DNA is not repaired properly, and cause increased the risk for breast cancer.[35,31]

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BRCA1 combines with other tumor suppressors, DNA damage sensors, and signal transducers to form a large multi-subunit protein complex known as the BRCA1-associated genome

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surveillance complex (BASC) [36] valosin-containing protein (VCP, also known as p97) plays a role to recruit BRCA1 to the damaged DNA sites. After ionizing radiation, VCP is recruited to

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DNA lesions and cooperates with the ubiquitin ligase RNF8 to orchestrate assembly of signaling complexes for efficient DSB repair.[37] BRCA1 interacts with VCP.[38] BRCA1 also interacts

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with c-Myc, and other proteins that are critical to maintain genome stability.[39] BRCA1 directly binds to DNA, with higher affinity for branched DNA structures. This ability to bind to DNA

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contributes to its ability to inhibit the nuclease activity of the MRNcomplex as well as the nuclease activity of Mre11 alone.[40] This may explain a role for BRCA1 to promote lower

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fidelity DNA repair by non-homologous end joining (NHEJ).[41] BRCA1 also colocalizes with γ-H2AX (histone H2AX phosphorylated on serine-139) in DNA double-strand break repair foci, indicating it may play a role in recruiting repair factors.[42] Formaldehyde and acetaldehyde are common environmental sources of DNA cross links that often require repairs mediated by BRCA1 containing pathways.[43] finally the importance of family history and pedigree- based screening is important for breast cancer screeing, as we know that family history also has impact in breast cancer screening.

4. ESTROGEN RECEPTORS

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Among the 17ß-estradiol target tissues uterus, mammary gland, placenta, liver, central nervous system, cardiovascular system and bone are the classical targets and these tissues have a high ER α content.[44] The nonclassical target tissues include prostate, testis, ovary, pineal gland, thyroid gland, parathyroids, adrenals, pancreas, gallbladder, skin, urinary tract and erythroid tissues where the expression of ER α is either very low or not measurable, whereas ERß is highly

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expressed.[45] ERs in the breast is their dual role in both proliferation and differentiation.[46] E2

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binds to transcription factors belonging to the nuclear receptor superfamily, in order to mediate

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its effect, the estrogen receptors (ER α and ERß), these are proteins encoded by 8 exons of 2 genes on different chromosomes.[47] ER encode proteins involved in apoptosis (as Bcl-2),

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cellular invasiveness (as cathepsin D), and cellular proliferation such as v-myc myelocytomatosis viral oncogene homolog (Myc) and transforming growth factor-alpha (TGFα). [48] On one hand,

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the expression of ERß is much lower in tumors than in healthy glands and in relation to ERα, ERß expression is reported to decrease during carcinogenesis.[49] Overexpression of ERß mRNA was reported in tumors from patients who became resistant to chemotherapeutics such as

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tamoxifen, where tamoxifen-liganded ERα and ERß have been shown to have strikingly different

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effects on activation number 1, AP-1 (a transcription factor) gene regulation with tamoxifenliganded ERα exerting antagonistic effects[50] expression of ER and mutant p53 is completely

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inverse in breast cancer cells.[51] ERα is capable of binding to MDM2 and the NH2 terminus of the p53 protein, thus protecting p53 from being deactivated by the MDM2 ligand

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independently.[52] It has been reported that ER-positive breast cancer cells have significantly higher (up to 30 folds) MDM2 mRNA levels than ER- negative ones, and this observed

[53]

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stimulatory effect of ERα on MDM2 was carried out via elevated p53 transcriptional activity.

4.1 HER2/neu HER2/neu is a proto-oncogene located on chromosome 17q11.2-q12, belongs to the epidermal growth factor receptor family.[54] It plays fundamental roles in development, proliferation,and differentiation. It is activated by its overexpression or transactivated by various ligands of EGF family.[55] The action of HER2/neu is by the formation of heterodimers with other ErbB receptors.[56] Ras/MAPK and PI3K/Akt are 2 downstream pathwaysof ErbB2, which link

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ErbB2 to its biological functions.[57] An amplification or over expression of tyrosine kinase receptor HER2/neu is found in approximately 20-40% of patients with breast cancer, as a result HER2/neu is overexpressed on the cell surface leading to increased oncogenesis.[58] There is evidence that over-expression of HER2 and p53 is involved in breast cancer progression, indicating the coexistence of HER2 over-expression and accumulation of p53protein is a strong

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prognostic molecular marker in breast cancer. [59,60].

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5. OTHER GENES INVOVED IN THE BREAST CANCER PROGRESSION

Rarer mutations also increase the risk of breast cancer as much as the BRCA genes. They are

ATM: The ATM gene normally helps repair damaged DNA. Inheriting 2 abnormal copies

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not frequent causes of inherited breast cancer.

of this gene causes the disease ataxia-telangiectasia. Inheriting 1 mutated copy of this 

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gene has been linked to a high rate of breast cancer in some families. TP53: The TP53 gene gives instructions for making a protein called p53 that helps stop

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the growth of abnormal cells. Inherited mutations of this gene cause Li-Fraumeni syndrome (named after the 2 researchers who first described it). People with this

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syndrome have an increased risk of developing breast cancer, as well as several other cancers such as leukemia, brain tumors, and sarcomas (cancer of bones or connective 

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tissue). This is a rare cause of breast cancer. CHEK2: The Li-Fraumeni syndrome can also be caused by inherited mutations in the

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CHEK2 gene. Even when it does not cause this syndrome, it can increase breast cancer risk about twofold when it is mutated. 

PTEN: The PTEN gene normally helps regulate cell growth. Inherited mutations in this gene can cause Cowden syndrome, a rare disorder in which people are at increased risk for both benign and malignant breast tumors, as well as growths in the digestive tract, thyroid, uterus, and ovaries. Defects in this gene can also cause a different syndrome called Bannayan-Riley-Ruvalcaba syndrome that is not thought to be linked to breast cancer risk. Recently, the syndromes caused by PTEN have been combined into one called PTEN Tumor Hamartoma Syndrome.

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

CDH1: Inherited mutations in this gene cause hereditary diffuse gastric cancer, a syndrome in which people develop a rare type of stomach cancer at an early age. Women with mutations in this gene also have an increased risk of invasive lobular breast cancer.

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STK11: Defects in this gene can lead to Peutz-Jeghers syndrome. People with this disorder develop pigmented spots on their lips and in their mouths, polyps in the urinary and gastrointestinal tracts, and have an increased risk of many types of cancer, including

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breast cancer.

PALB2: The PALB2 gene makes a protein that interacts with the protein made by the

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BRCA2 gene. Defects (mutations) in this gene can lead to an increased risk of breast cancer. It isn’t yet clear if PALB2 gene mutations also increase the risk for ovarian

MOLECULAR PATHWAYS

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6. BREAST CANCER PATHOGENESIS

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cancer and male breast cancer.

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Potential elements observed in breast cancer are DNA amplification (proto -oncogenes, growth factors and their receptors) and DNA deletion (in tumorsuppressor genes). Berouk him et al

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found 76 amplifications and 82 deletions in 243 breast tumors, in regions containing new possible sensitive genes, such as MCL1 and BCL2L1 (apoptosis), Interleukin-1 receptor-

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associated kinase1 (IRAK1), TNF receptor associated factor (TRAF) 6, IKBKG which codes NF-kappa-B essential modulator (NEMO) protein and IKBKB which codes inhibitor of nuclear

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factor kappa-B kinase subunit beta (IKK-B) protein in NK- kB signaling pathway. PIK 3CA, the gene encoding the catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is mutated in about 20 – 30% of breast tumors. TP53 mutations are found in about 30 – 35% of cases.[61]

6.2 TUMOR SUPRESSOR GENES

BRCA1 (Breast Cancer gene A1) and BRCA 2 (Breast Cancer gene A2), TP53 mutations.[61] are tumor suppressor genes associated with breast cancer. Mostly patients with mutation in BRCA1 usually bear triple-negative kind breast tumors . Insulin-like Growth Factor (IGF) may

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have effect in breast cancer progression. It has been showed that Retinoic Acid (RA) mediate their inhibitory effects on cell growth of cancerous human breast cancer cells “MCF7” via selective reduction of Insulin Receptor Subtype-1 (IRS-1) and its activity which results in the selective downregulation of IP3-kinase/AKT. High levels of Irs-1 in human breast tumors correlate with elevated incidence of disease recurrence. Although the insulin receptor substrates (IRS) were primarily identified, as the name implied, as a substrate for the insulin receptor

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(IR),Nowadays it has been known that these adapter proteins, are involved in activation of

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downstream pathways of several growth factor receptors such as insulin-like growth factor- 1

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receptor (IGF-1R), vascular endothelial growth factor receptor (VEGF-R), cytokine receptors, and some members of the integrin family. Loss of either IRS -1 or IRS -2 did not show similar

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consequence on developing lung metastasis. Metastasis will increase in IRS -1-deficient tumors, IRS-2-deficient tumors shows decreased lung metastasis. It is thought that a compensatory

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mechanism which upregulate IRS-2 expression is involved in the increased metastasis seen in IRS-1-deficient tumors.

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Akt1 was shown to inhibit invasion and metastasis while Akt2 perform in an opposite way. RA influence occurs at post-translational level byincrease in ubiquitination and serine

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phosphorylation of IRS-1. The latter is protein-kinase C (PKC)-dependent, since PKC inhibitors block the process. Activation of PKC-delta by RA hasalso been reported. Activation of

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PI3K/PDK/Akt cascade also decreases sensitivity of MCF7 cells to anticancer drugs. Induction

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of Bcl-2 may contribute to this resistance.

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6.3 ASTROCYTE ELEVATED GENE-1(AEG-1)

Astrocyte elevated gene-1(AEG-1, also known as Metadherin and lyric) elevation in human breast cancer causes leads to enhanced cell proliferation and their ability of anchorageindependent growth of breast cancer cells. The attenuation of two key cell-cycle inhibitors, p27Kip1 and p21Cip1, via Akt/FOXO1 signaling pathway causes the proliferative effects. FOXO1 is a transcription factor belonging to the Forkhead box-containing class O (FOXO) subfamily. Many biological functions have been shown to be related with FOXO1 including cellcycle control, differentiation, stress response and apoptosis. [62] FOXO proteins could act as

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tumor suppressors through induction of CDK inhibitors, including p21 Cip1, p27Kip1 and p57.[63] Overexpression of AEG-1 increases migration and invasion of human glioma cells because of the presence of a lung-homing domain which facilitates breast tumor metastasis to lungs. Recent observations indicate that AEG-1 play this role by activating NF-κB pathway. AEG-1 facilitates IκBa degradation, resulting in an increase in NF- κB DNA binding activity and NF- κB promoter activity in reporter assays These valuable findings are strengthen the idea

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which recommend AEG-1 as a crucial regulator of tumor progression and metastasis.[64] AEG-1

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is mediating a broad-spectrum chemoresistance by the pro survival pathways such as PI3K and

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NFκ B, or through other downstream genes of MTDH/AEG-1 that directly regulate

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chemoresistance.[65]

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6.4 miRNAs

Oncogenic miRNAs acts by suppressing tumor suppressor genes. Some of the miRNAs exhibit

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tumor suppressor properties by down-regulating oncogenic genes.[66] Downregulation of angiogenesis is caused by oncogenic miRNAs . Expression of miR-126 is upregulated in breast

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cancer cells.[67]] By inhibiting the protein synthesis of insulin-like growth factor binding protein 2, c-Mer tyrosine kinase and phosphatidylinositol transfer protein cytoplasmic 1, miR-126

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affects angiogenesis . miRNAs 10b and 196b regulate angiogenesis targeting vascular endothelial growth factor (VEGF) signaling through HOXD10 miRNAs are known to inhibit

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tumor suppressor genes by affecting epigenetic changes. In breast cancer cells MDA-MB-231 and MCF-7 miRNAs miR-7 and miR-218 affects histone modification and DNA methylation by

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targeting HOXB3. This results in inhibition of RASSF1A and Claudin 6 expression.[68]

7. SIGNAL PATHWAYS

Tumorogenensis occurs by dysregulation of signal transduction pathways due to mutations. Increased expression of specific receptor tyrosine kinases (RTKs) has been implicated in the genesis of a significant proportion of sporadic human breast cancers. Increased activity of tyrosine kinases can result in aberrant cell proliferation. This phenomenon may result in cel transformation. For example, amplification and overexpression of neu/erbB2 protooncogene is

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observed in 20–30% human breast cancer, and is inversely correlated with the survival of the patient. Epidermal growth factor receptor (EGFR) family is a member of growth factor receptors which consists of four members: EGFR, ErbB2/Neu, ErbB 3, and ErbB 4. Increase ErbB2 expression, has been further associated with poor clinical outcome, is observed in 20 – 30% of sporadic breast tumors. The main reason is ErbB2 gene amplification.[69] Increased level of tyrosine phosphorylated ErbB3 is reported. ErbB3 is a bridge which links the phosphatidyl

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inositol-3 kinase (PI-3K) signaling molecule to Neu which has attracted much attention because

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of its potent transforming properties. This oncogene activates a number of common signaling

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pathways by providing specific binding sites for a variety of signaling molecules that include either Src Homology 2 (SH2) or phosphotyrosine binding/interacting domains.[70] Co-

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expression of ErbB2 and ErbB3 RTKs is usually observed in common tumor progression.[69,71] Polyoma virus middle T (PyV mT) antigen, another tyrosine kinase involved in murine

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mammary tumorigenesis and metastasis, results in the rapid induction of multifocal metastatic mammary tumors. Since these tumors occur during early steps of mammary gland development

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and involve whole of the gland, expression of PyV mT will result in transformation of the primary mammary epithelium. This molecule is also associated with many signaling pathways

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via Src Homology 2 (SH2) or phosphotyrosine binding/interacting domains.[70] Induction of tumor by the PyV M T oncogene is also dependent on the presence of functional ß1-integrin.

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Lack of functional ß1-integrin makes tumor cells unable to enter the cell cycle. Tumor cells are unable to proliferate, there are still viable and bears pathological tumor dormancy.[69]

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Phosphatidyl inositol-3 kinase is also important in mammary tumor progression. Association of PI-3K links to PyV mT through its binding to phosphotyrosine residues (Tyr 315/322) within the

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PyV mT coding sequences. Association with Neu happens through recruitment to ErbB3 (ErbB, is derived from the name of a viral oncogene to which these receptors are homologous: Erythroblastic Leukemia Viral Oncogene). Activation of PI-3K and resultant production of phosphoinotide-3 lipids stimulates several members of serine kinase family. The final of these cascades will be the stimulation a number of antiapoptotic signaling molecules such as nuclear factor-kB (NF- κB)[72,73] 7.1 NF- κ B

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NF- κ B has regulatory role on the expression of various tumor-promoting molecules such as MMP, cycloxygenase 2, inducible nitric oxide synthase, chemokines, and inflammatory cytokines explain its significant effect on bearing cancer. NF- κB increased the expression of these molecules, all of which enhance tumoral cell invasion and angiogenesis. Other aspect of the role of NF- κB in tumorigeneses includes increasing expression of protooncogenes such as c-

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myc and cyclin D1 which directly stimulate proliferation.[72]

regulate protein – protein interaction and help the formation of protein

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Adapter proteins

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7.2 ADAPTER PROTEINS

complex which participate in signal transduction pathways. They do not exert any kinase

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activity. GRB2-associated-binding protein 2 (Gab2) is one of the adapter proteins which is overexpressed in breast cancer. It promotes signaling pathways by recruiting SH2 containing

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proteins such as PI3K, Shc, and Shp2 downstream of tyrosine kinase receptors. Elevated expression of Gab2 in the mammary epithelium is unable to induce tumor development, it has

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been shown that tumor onset time will decrease in presence of Gab2.[74,75]

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7.3 RAS PATHWAY

Mammary tumor progression is associated with the activation of Ras signaling pathway and

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adapter proteins such as Shc and Grb2 create some specific complexes with activated forms of Neu and PyV mT. The co-operation of Grb2 and Shc with these activated oncoproteins will

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result in stimulation of Ras signaling. In contrast to PyV mT, which signals to Ras only through its association with Shc, Neu can activate Ras through Grb2, Shc and several other unidentified

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adapter proteins. Ras activation will be the recruiting of a number of downstream effector molecules including PI-3K, Raf serine kinase, GRB associated-binding protein (GAP) and Rasrelated protein (Ral).[74,75]

7.4 MAPK Mitogen-activated protein kinases (MAPK) are a family of Ser/Thr protein kinases in eukaryotes involved in many cellular programs such as cell proliferation, cell differentiation, cell movement, and cell death. MAPK signaling cascades are organized hierarchically into three-tiered modules. MAPKs are phosphorylated and activated by MAPK-kinases (MAPKKs), which in turnare

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phosphorylated and activated by MAPKK-kinases (MAPKKKs). The MAPKKKs are inturn activated by interaction with the family of small GTPases and/or other protein kinases, connecting the MAPK module to cell surface receptors or external stimuli.

7.5 CELL CYCLE ALTERATION Cyclin D1is overexpressed in human breast cancer. Overexpression of Cdc25b make mammary

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glands hyperplasic and more sensitive to carcinogenic chemicals.It does not directly induce

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tumorigeneses. Inhibitor of nuclear factor kappa-B kinase (IKK a, a responsible kinase for

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activation of NF-k B, was identified as a necessary factor for Cyclin D1-associated epithelial

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proliferation in MMTV-Neu (but not in MMTV- Ra s) mice.[76]

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8. STEM CELLS HYPOTHESIS

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Cancer stem cells (CSC) hypothesis explains that tissue specific Stem Cells (SCs) and their early progenitors as the main causes of the malignant behaviour of cancer. Stem cells are

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undifferentiated and have the ability to divide into two daughter cells. Division is asymmetrical and will cause an identical clone of the mother cell and another cell which can divide and fully

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differentiate into new cell line. This latter daughter cell is named a Progenitor. Breast SCs are very long life and thus influences of the effect of chemicals and radiation. Breast CSCs escape

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from the control of surrounding micro environment, they are able to bear malignant progenitor offspring. The result will be the production of malignant daughter cells that create the bulk of the spared by current cancer therapies in

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tumour. Some of the breast CSCs are quiescent and are which rapidly dividing cells are targeted.[77-80]

9. STAT FAMILY: (signal transducer and activator of transcription)

STAT family of proteins are latent cytoplasmic transcription factors which are involved in cytokines signaling pathways. STAT proteins need activation through tyrosine phosphorylation, which leads to dimerization via conserved structural features phosphotyrosine-SH2 (Src

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homology domain 2) of two Stat molecules. Fallowing activatin, Stats transport to the nucleus, where they bind to the Activatin of STAT3 happens by recruitment to phosphotyrosine motifs within complexes of growth factor receptors (e.g.,epidermal growth factor receptor), cytokine receptors (e.g., IL-6 receptor), or non-receptor tyrosine kinases (e.g., Src and BCR-ABL) through their SH2 domain. Stat3 is phosphorylated on a tyrosine residue by activated tyrosine kinases in receptor complexes. Phosphorylated Stat3 forms homodimers and heterodimers and

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translocates to the nucleus.In the nucleus, Stat3 dimers bind to specific promoter elements of

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target genes and regulate gene expression. The Stat3 signaling pathway regulates cancer

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metastasis by regulating the expression of genes that are critical to cell survival, cell proliferation, invasion, angiogenesis and tumor immune evasion. promoter of target genes and

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activate their transcription. Dimerized status of STATs istransient in normal non-transformed cells. But in transformed cancerous cells, Stat proteinsin particular, Stat3 are found in a

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permanent active dimerized manner. Activated form of STAT3 has been found in more than 50% of primary breast tumors and tumor-derived celllines. It has been reported that expression of a

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constitutively active form of Stat3 (Stat3C) is sufficient for promoting cellular transformation and bearing an immortalized breast cellline. Since the IL-6/gp130/Jak signaling pathway has a

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crucial role in Stat3 activation inhuman breast cancer, blockade of this pathway may be an

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important therapeutic plan in breast cancer therapy.[83]

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10. ASSOCIATED MALIGNANCY

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11.1 HEREDITARY BREAST AND OVARIAN CANCER

Genetic mutations of the BRCA1 and BRCA2 genes cause the Hereditary breast-ovarian cancer syndrome (HBOC). It is an autosomal dominant genetic disorder. In women this disorder primarily increases the risk of breast and ovarian cancer and also increases the risk of fallopian tube carcinoma and papillary serous carcinoma of the peritoneum. In men the risk of prostate cancer is increased. The other cancers that are linked to this syndrome are pancreatic cancer, male breast cancer, colorectal cancer and cancers of the uterus and cervix. Genetic mutations account for approximately 7% and 14% of breast and ovarian cancer, respectively, and BRCA1 and BRCA2 account for 80% of these cases.[84,85]

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The mutations can be changes in one or a small number of DNA base pairs (the building-blocks of DNA) or large segments of DNA are rearranged. These mutations can be identified with PCR and DNA sequencing. Those large segments, also called large rearrangements, can be a deletion or a duplication of one or several exons in the gene. Hypermethylation of the BRCA1 promoter, which has been reported in some cancers, could be considered as an inactivating mechanism for BRCA1 expression.[86] Classical methods for mutations detection (sequencing) are unable to

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reveal those mutations.[87] Hence other methods like quantitative PCR,[88] Multiplex

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Ligation-dependent Probe Amplification (MLPA),[89] and Quantitative Multiplex PCR of Shorts

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Fluorescents Fragments (QMPSF)[90] need to be used. New methods like heteroduplex analysis (HDA) by multi-capillary electrophoresis or also dedicated oligonucleotides array based

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on comparative genomic hybridization (array-CGH).[91] A mutated BRCA1 gene usually makes a protein that does not function properly. Researchers believe that the defective BRCA1 protein

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is unable to help fix DNA damages leading to mutations in other genes. These mutations can accumulate and may allow cells to grow and divide uncontrollably to form a tumor. Thus,

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BRCA1 inactivating mutations lead to a predisposition for cancer. BRCA1 mRNA 3' UTR can be bound by an miRNA, Mir-17 microRNA. It has been suggested

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that variations in this miRNA along with Mir-30 microRNA could confer susceptibility to breast cancer.[92]

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In addition to breast cancer, mutations in the BRCA1 gene also increase the risk of ovarian, fallopian tube, and prostate cancers. Moreover, precancerous lesions (dysplasia)

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within the Fallopian tube have been linked to BRCA1 gene mutations. Pathogenic mutations anywhere in a model pathway containing BRCA1 and BRCA2 greatly increase risks for a subset

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of leukemias and lymphomas.

11.2 LI-FRAUMENI SYNDROME

Li-Fraumeni syndrome is associated with the development of a number of cancers, including breast cancer sarcoma (such as osteoscarcoma and soft-tissue sarcomas), leukemia, brain (central nervous system) cancers and cancer of the adrenal cortex . The cancers most often occur in childhood, although the breast cancers occur in young adults. People with Li-Fraumeni can also be affected by more than one cancer in their lifetime. This rare syndrome is at higher risk

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of cancer from radiation therapy. It is very important to avoid giving radiation when treating these patients.[93]

11. BREAST CANCER TYPES AND STAGES

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12.1 TYPES OF BREAST CANCER:

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Breast cancer is typed as invasive or noninvasive (often referred to as in situ). Noninvasive

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breast cancers is of two types 1) ductal carcinoma in situ (DCIS) and 2) lobular carcinoma in situ (LCIS). The noninvasive breast cancers do not invade the basement membrane of the

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breast. Ductal carcinoma in situ cancer cells are found in the lining of the duct and whereas lobular carcinoma in situ cancer cells are found in the lobules. Invasive breast cancer is of two

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types, 1) infiltrating ductal carcinoma and 2) infiltrating lobular carcinoma. Infiltrating ductal carcinoma penetrates the wall of the duct and travels to areas outside of it. Infiltrating lobular

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carcinoma spreads through the wall of the lobule and travels to areas outside of it. Infiltrating ductal carcinoma is the most common type of breast cancer, accounting for between 70%-80% of

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the cases of breast cancer.

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12.2 STAGES OF BREAST CANCER:

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Each of the four types of breast cancer has four stages that relate to the severity of the cancer. Stage 0— non-invasive

carcinomas (LCIS or DCIS). Cancer cells have not invaded the

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surrounding breast tissue.

Stage I —the tumor is no more than 2 cm in size and cancer cells have not spread beyond the breast.

Stage II—either the tumor has spread to the lymph nodes under the arms but the tumor is less than2cm. in size, or the tumor has not spread to the lymph nodes under the arms but is greater than 5 cm in size, or the tumor is between 2 and 5 cm and may or may not have spread to the nodes. Stage III—the tumor is greater than 5 cm in size and has spread to the lymph nodes under the arms.

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Stage IV—the cancer has spread to other parts of the body (metastatic cancer).

12.2 STAGING OF BREAST CANCER FOR THERAPEUTIC PURPOSE:

Estrogen receptor (ER)-negative tumors and basal-like and human epidermal growth factor receptor-2 (HER2)-enriched, and two subtypes of ER-positive tumors including luminal A and

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luminal B. These subtypes differ markedly in the luminal cancers, luminal A and luminal B, so

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called because they are characterized by expression of genes also expressed by normal breast

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luminal epithelial cells, have overlap with ER-positive breast cancers. There are also several subtypes characterized by low expression of hormone receptor-related genes (ER-negative), one

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of which is called the "HER2-enriched" subtype (previously called HER2+/ER-) and another called the "basal-like"subtype. The basal-like subtype is named because it expresses many genes

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characteristic of normal breast basal epithelial cells. 12.3 LUMINAL SUBTYPES

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The name "luminal" derives from similarity in expression between these tumors and the luminal epithelium of the breast; they typically express luminal cytokeratins 8 and 18. These are the most

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common subtypes, make up the majority of ER-positive breast cancer, and are characterized by

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expression of ER, PR, and other genes associated with ER activation.

12.4 LUMINAL A AND LUMINAL B TRAITS

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High expression of ER-related genes, low expression of the HER2 cluster of genes, and low expression of proliferation-related genes are the two main characters of Luminal A tumors. This

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kind has the best prognosis of all breast cancer subtypes. Whereas luminal B tumors have relatively lower (although still present) expression of ER-related genes, variable expression of the HER2 cluster, and higher expression of the proliferation cluster.Luminal B tumors carry a worse prognosis than luminal A tumors. Unfortunately, this subtype has high probability of recurrence.

12.5 HER2-ENRICHED SUBTYPE The HER2-enriched subtype (previously the HER2+/ER- subtype) is characterized by high expression of the HER2 and proliferation gene clusters, and low expression of the luminal

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cluster. For this reason, these tumors are typically negative for ER and PR, and positive for HER2. It is important to note that this subtype comprises only about half of clinically HER2positive breast cancer. The rest have high expression of both the HER2 and luminal gene clusters and fall in a luminal subtype. Promotion in HER2-directed therapy has improved the poor prognosis of this subtype.

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12.6 BASAL-LIKE SUBTYPE

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The name of “basal-like” subtype comes from the similarity in gene expression to that of the

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basal epithelial cells. This subtype shows lower expression of the luminal and HER2 gene clusters. Therefore, these tumors are typically ER-, PR-, and HER2-negative on clinical assays.

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Because of this reason, the name "triple negative" is also used to describe them. However, while most triple negative tumors are basal-like, and most basal-like tumors are triple negative, there is

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significant inconsistency (up to 30 percent) between these two classifications. Although any subtype can be triple negative on clinical assays, an interesting subtype found in non-basal triple

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negative breast cancers is the more newly described claudin-low subtype, which is uncommon but interesting because of its expression of epithelial-mesenchymal transition genes and

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characteristics reminiscent of stem cells .

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12.7 TRIPLE-NEGATIVE BREAST CANCER Triple-negative breast cancer (TNBC) comprises 10 % to 20 % of breast cancers and is

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characterized by a lack of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor-2 (HER2) expression. TNBCs are usually composed of

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biologically aggressive and histologically high-grade tumors and tend to relapse within 3 years of diagnosis with the wors clinical outcome at this juncture.[94,95] TNBCs with non-pCR have been reported to be associated with a markedly worse prognosis.[96] Oncogenic Ha-Ras increases MTDH/AEG-1 expression through the activation of the PI3K/Akt pathway, which phosphorylates and inactivates GSK3β, and subsequently enhances the stabilization and binding of c-Myc to the MTDH/AEG-1 promoter. MTDH/AEG-1 can activate AKT, NFκB, and Wnt/β-catenin pathways to promote proliferation, survival, and invasion. Activation of NFκB signaling is in part mediated by the direct interaction of MTDH/AEG-1 with p65 and CBP, a generaltranscriptional co-activator. MTDH/AEG-1 activates the Wnt/β-catenin

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pathway through increasing the activity of MAPK kinases ERK and p38, which phosphorylates GSK3β and stabilized β-catenin. Furthermore, MTDH/AEG-1 increases the expression of LEF-1, a transcriptional cofactor for β-catenin. The pro metastasis function of MTDH/AEG-1 is mediated by the interaction of the LHD of MTDH/AEG-1 with an unknown receptor in endothelial cells. The broad spectrum chemoresistance function of MTDH/AEG-1 is mediated by a number of downstream genes that promote the resistance to multiple chemotherapeutic agents.

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Proteins with direct interactions with MTDH/AEG1 are shown in green. Figure.2 shows that

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mtdh/aeg-1 promotes tumor progression through the integration of multiple signaling

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pathways.[103]

Activated protein C (APC), an anticoagulant serine protease, is related to cell survival, cell

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migration, angiogenesis and breast cancer invasion. APC recruits EPCR, PAR-1, and EGFR in extracellular matrix in order to increase the invasive properties of MDA-MB-231 cells. Other

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mechanisms include activation of matrix metalloprotease (MMP) -2 and/or -9 and activation of ERK, Akt, and NF-κB (but not the JNK) pathways. APC does not employ the endogenous

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plasminogen activation system to increase invasion. [104]. Figure.3 shows the role of apc in

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breast cancer progressions[104]

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12. ADHESION MOLECULES

Increased metastatic properties of breast cancer occurs by dysregulation of protein expression.

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Reduction in cell adhesion and increased cell motility is necessary for tumor metastasis. Adhesion molecules including selectins, integrins, lectins, and cadherins are associated with

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metastasis. [105-109] The cells have to pass the basement membrane to reach the surrounding vessels and spread to other sites. The process involves proteolysis and motility and need proteolytic enzymes to work. Three major categories of proteolytic enzymes including the matrix metalloproteinases [110] serine proteinases, and cathepsins are implicated in metastasis. Cell motility is another factor which cells need to be able to metastasize to other tissues. Several factors are necessary for cellular motility, including the autocrine motility factor, autotaxin, and hepatocyte growth factor (HGF). HGF leads to metastases. 13. BONE METASTASIS

more as well as larger axillary lymph node

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Osteonectin engages breast and prostate cancer cells to bone. Osteonectin is a glycoprotein secreted by osteoblasts in bone, initiating mineralization and promoting mineral crystal formation. Chemokine receptors CXCR4 and CCR7 express in breast carcinoma cells predisposed for metastasis to lymph nodes and bone. MTA1 is found in the chromatin remodeling histone deacetylase complex.Metastatic potential is propotional to Metastasisassociated protein 1 (MTA1) mRNA expression. Osteopontin is a metastasis associated gene.

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Elevated plasma levels of Osteopontin and immune histochemical staining of tumor cells are

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found in metastatic breast cancer patients.[111]

14. METASTASIS SUPPRESSOR GENES

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15.1 E-cadherin

E-cadherin has been demonstrated to correlates negatively with the potential of tumor invasion.

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It is a member of the cadherin superfamily of Ca2+-dependent adhesion cell surface molecules, expressed predominantly in epithelial tissues Reduction and/or loss of E-cadherin expression in

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carcinomas will result in increased tumor metastasis because of the reduction in tumor cell adhesiveness and increased cell motility.[112]

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15.2 Tissue Inhibitors of Metalloproteinases[TIMS] Metalloproteinases suppress tumor metastasis.The role of metalloproteinases (TIMPs) is

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inhibiting the activity of matrix proteinases (MMPs). Increased TIMPs are associated with progression to metastatic disease. One proposed explanation is that the balance between MMPs

15.3 Maspin

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and TIMPs is important than the expression of each protein.[113]

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Maspin is a tumor suppressor gene which has been established to be involved at least in breast and prostate cancer. Maspin is belonging to the serpin family of serine protease inhibitors.[114] 15.4 Kai1

Kai1 is a member of the Transmembrane-4 super family of adhesion molecules and is involved in lymphocyte differentiation and function. Kangai 1 in Chinese kang ai meaning anticancer. It was originally described as a metastasis suppressor in prostate cancer but its role has been established as a general suppressor of the metastatic phenotype in many cancer types including breast cancer.[115] 15.5 BRMS1

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Breast cancer metastasis-suppressor 1 (BRMS1) decreases metastatic potential of tumour cells. The tumour suppression by BRMS1 is mediated by enhanced immune recognition, altered transport, and/or secretion of metastasis-associated proteins.[116] 15.6 MKK4 MKK4 belongs to the Ser/Thr protein kinase family. This gene encodes a dual specificity protein kinase. MKK4 is a direct activator of MAP kinases in response to various environmental stresses

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or mitogenic stimuli. It activates MAPK8/JNK1, MAPK9/JNK2, and MAPK14/p38, but not

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MAPK1/ERK2 or MAPK3/ERK3. This kinase is phosphorylated, and thus activated by

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MAP3K1/MEKK.[117]

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15. DIAGNOSIS

cancer have no symptoms. Hence

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Breast cancer is sometimes found after symptoms appear, but many women with early breast recommended the

screening tests.These might include

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imaging tests, looking at samples of nipple discharge, or doing biopsies of suspicious areas. Imaging tests used to evaluate breast disease Imaging tests use x-rays, magnetic fields, sound

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waves, or radioactive substances to create pictures of the inside of your body. 16.1 MAMMOGRAMS

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A mammogram is an x-ray of the breast. Screening mammograms are used to look for breast disease in women who have no signs or symptoms of a breast problem. Screening mammograms

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usually take 2 views (x-ray pictures taken from different angles) of each breast. 16.2 BREAST ULTRASOUND

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Ultrasound, also known as sonography, uses sound waves to outline a part of the body. For this test, a small, microphone-like instrument called a transducer is placed on the skin (which is often first lubricated with ultrasound gel). It emits sound waves and picks up the echoes as they bounce off body tissues. The echoes are converted by a computer into a black and white image that is displayed on a computer screen. This test is painless and does not expose you to radiation.Ultrasound has become a valuable tool to use along with mammography because it is widely available and less expensive than other options, such as MRI. Usually, breast ultrasound is used to target a specific area of concern found on the mammogram. Ultrasound helps distinguish between cysts (fluid-filled sacs) and solid masses and sometimes can help tell the

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difference between benign and cancerous tumors. In someone with a breast tumor, it can also be used to look for enlarged lymph nodes under the arm.The use of ultrasound instead of mammograms for breast cancer screening is not recommended. However, clinical trials are now looking at the benefits and risks of adding breast ultrasound to screening mammograms in women with dense breasts and a higher risk of breast cancer. 16.3 MAGNETIC RESONANCE IMAGING (MRI) OF THE BREAST:

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MRI scans use radio waves and strong magnets instead of x-rays. The energy from the radio

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waves is absorbed and then released in a pattern formed by the type of body tissue and by certain

vein before or during the scan to show details better.

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16.4 NIPPLE DISCHARGE EXAM

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diseases. For breast MRI to look for cancer, a contrast liquid called gadolinium is injected into a

Nipple discharge fluid may be collected and looked at under a microscope to see if any cancer

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cells are in it. Most nipple discharges or secretions are not cancer. In general, if the secretion appears milky or clear green, cancer is very unlikely. If the discharge is red or red-brown,

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suggesting that it contains blood, it might possibly be caused by cancer, although an injury, infection, or benign tumors are more likely causes.Even when no cancer cells are found in a

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nipple discharge, doctors cannot be sure breast cancer is not present. 16.5 DUCTAL LAVAGE AND NIPPLE ASPIRATION

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Ductal lavage is an experimental test developed for women who have no symptoms of breast cancer but are at very high risk for the disease. It is not a test to screen for or diagnose breast

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cancer, but it may help give a more accurate picture of a woman's risk of developing it. An anaesthetic cream is applied to numb the nipple area. Gentle suction is then used to help draw

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tiny amounts of fluid from the milk ducts up to the nipple surface, which helps locate the ducts' natural openings. A tiny tube (called a catheter) is then inserted into a duct opening. Saline (salt water) is slowly infused into the catheter to gently rinse the duct and collect cells. The ductal fluid is drawn through the catheter and sent to a lab, where the cells are looked at under a microscope. Nipple aspiration also looks for abnormal cells developing in the ducts, but is much simpler, because nothing is inserted into the breast. The device for nipple aspiration uses small cups that are placed on the woman's breasts. The device warms the breasts, gently compresses them, and applies light suction to bring nipple fluid to the surface of the breast. The nipple fluid is then

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collected and sent to a lab for analysis. As with ductal lavage, the procedure may be useful as a test of cancer risk but is not an appropriate screening test for cancer. The test has not been shown to detect cancer early. 16. BIOPSY

A biopsy is done when mammograms, other imaging tests, or the physical exam finds a breast

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change (or abnormality) that is possibly cancer. A biopsy is the only way to tell if cancer is really

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present. There are several types of biopsies, such as fine needle aspiration biopsy, core (large

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needle) biopsy, and surgical biopsy. Each has its pros and cons. The choice of which to use depends on your specific situation.

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17.1 FINE NEEDLE ASPIRATION BIOPSY:

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In a fine needle aspiration (FNA) biopsy, hollow needle attached to a syringe to withdraw (aspirate) a small amount of tissue from a suspicious area, which is then looked at under a

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microscope. The needle used for an FNA biopsy is thinner than the one used for blood tests. If the area to be biopsied can be felt, the needle can be guided into the area of the breast change

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while the doctor is feeling (palpating) it.If the lump can't be felt easily, the doctor might use ultrasound to watch the needle on a screen as it moves toward and into the mass. A local

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anesthetic (numbing medicine) may or may not be used. Because such a thin needle is used for the biopsy, the process of getting the anesthetic may actually be more uncomfortable than the

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biopsy itself. Once the needle is in place, fluid is drawn out. If the fluid is clear, the lump is probably a benign cyst. Bloody or cloudy fluid can mean either a benign cyst or, very rarely, a

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cancer. If the lump is solid, small tissue fragments are drawn out. A pathologist will look at the biopsy tissue or fluid under a microscope to determine if it is cancerous. An FNA biopsy is the easiest type of biopsy to have, but it has some disadvantages. It can sometimes miss a cancer if the needle is not placed among the cancer cells. And even if cancer cells are found, it is usually not possible to determine if the cancer is invasive. In some cases there may not be enough cells to perform some of the other lab tests that are routinely done on breast cancer specimens. If the FNA biopsy does not provide a clear diagnosis, or your doctor is still suspicious, a second biopsy or a different type of biopsy should be done.

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17.2 CORE NEEDLE BIOPSY A core biopsy uses a larger needle to sample breast pinpointed by ultrasound or mammogram. When mammograms taken from different angles are used to pinpoint the biopsy site, this is known as a stereotactic core needle biopsy.In some centers, the biopsy can be guided by an MRI scan. The needle used in core biopsies is larger than the one used in FNA. It removes a small cylinder (core) of tissue (about 1/16- to 1/8-inch in diameter and ½-inch long) from a breast

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abnormality. Several cores are often removed. The biopsy is done using local anesthesia (you are

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awake but the area is numbed) in an outpatient setting. Because it removes larger pieces of

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tissue, a core needle biopsy is more likely than an FNA to provide a clear diagnosis, although it

17.3 VACUUM-ASSISTED CORE BIOPSIES

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might still miss some cancers.

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Another way to do a core biopsy is known as vacuum-assisted. For this procedure, the skin is numbed and a small incision (about ¼ inch) is made. A hollow probe is inserted through the

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incision into the abnormal area of breast tissue. The probe is guided into place using mammography, ultrasound, or MRI. A cylinder of tissue is then suctioned in through a hole in

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the side of the probe, and a rotating knife within the probe cuts the tissue sample from the rest of the breast. Several samples can be taken from the same incision. Vacuum-assisted biopsies are

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done as an outpatient procedure. No stitches are needed, and there is minimal scarring. This

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method usually removes more tissue than a regular core biopsy.

17.4 SURGICAL (OPEN) BIOPSY

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Usually, breast cancer can be diagnosed using needle biopsy. Rarely, surgery is needed to remove all or part of the lump for microscopic examination. This is referred to as a surgical biopsy or an open biopsy. Most often, the surgeon removes the entire mass or abnormal area as well as a surrounding margin of normal-appearing breast tissue. This is called an excisional biopsy. If the mass is too large to be removed easily, only part of it may be removed. This is called an incisional biopsy.

This type of biopsy can also be done under general anesthesia. If the breast change cannot be felt, a mammogram may be used to place a wire into the correct area to guide the surgeon. This

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technique is called wire localization or stereotactic wire localization. After the area is numbed with local anesthetic, a thin hollow needle is placed in the breast, and x-ray views are used to guide the needle to the suspicious area. Once the tip of the needle is in the right spot, a thin wire is inserted through the center of the needle. A small hook at the end of the wire keeps it in place. The hollow needle is then removed. The surgeon can then use the wire as a guide to the abnormal area to be removed. The surgical specimen is sent to the lab to be looked at under a

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microscope. A surgical biopsy is more involved than an FNA biopsy or a core needle biopsy. It

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typically requires several stitches and may leave a scar. The larger the amount of tissue removed,

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the more likely it is that you will notice a change in the shape of your breast afterward. Core needle biopsy is usually enough to make a diagnosis, but sometimes an open biopsy may be

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needed depending on where the lesion is, or if a core biopsy is not conclusive. All biopsies can cause bleeding and can lead to swelling. This can make it seem like the breast lump is larger

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after the biopsy. This is generally nothing to worry about and the bleeding and bruising resolve

17.5 LYMPH NODE BIOPSY

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quickly in most cases.

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If the lymph nodes under the arm are enlarged (either when felt or on an imaging test like mammography or ultrasound), they may be checked for cancer spread. Most often, a needle

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biopsy is done at the time of the needle biopsy of the breast tumor. Even if no lymph nodes are enlarged, the lymph nodes under the arm are usually checked for cancer spread when the breast

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tumor is removed at surgery. This is done with a sentinel lymph node biopsy and/or an axillary

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lymph node dissection. [118,119]

17. MITOCHONDRIAL MARKERS

Mitochondria DNA (mtDNA) is present at 1000-10,000 copies/cell, and the vast majority of these copies are identical (homoplasmic) at birth. Mitochondria typically contain multiple haploid copies of their own genome (16.5 kb), including most components of transcription, translation, and protein assembly. Several mutations in the mtDNA, particularly in the D-loop region have been recently found in breast, colon, oesophageal, endometrial, head and neck, liver,

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kidney, leukemia, lung, melanoma, oral, prostate, and thyroid cancer.[130] The majority of these somatic mutations are homoplasmic in nature, suggesting that the mutant mtDNA played an active role in tumour formation.[131] By virtue of their clonal nature and high copy numbers in cancer cells, mitochondrial mutations may provide a powerful molecular marker for non-invasive detection of cancer. It may also be useful in early detection, diagnosis, and prognosis of cancer outcome and/or in monitoring response to certain preventive and interventional modalities as

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well as therapies.[132,133] Mutated mtDNA has also been detected in the body fluids of cancer

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patients and indeed is much more abundant than the mutated nuclear p53 DNA.[134] Since the

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mitochondrial gene expression signatures of transformed cells have now been identified, development of mitochondrial functional proteomics is expected to identify new markers for

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early detection and risk assessment, as well as targets for therapeutic intervention.[135] 18. METABOLIC BIOMARKER (GLUCOSE METABOLISM)

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Warburg observed that enhanced glucose utilization is a prominent and fundamental change in many tumours irrespective of their histological origin and the nature of mutations.(136,137] The

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fundamental alteration in metabolism during carcinogenesis includes mutations in the

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mitochondrial DNA resulting in functional impairment, oncogenic transformation linked upregulation of glycolysis, enhanced expression of metabolic enzymes and adaptation to the hypoxic tumour micro-milieu in case of solid tumours.[138] Bio-energetic index of the cell (BEC

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index) has been suggested that could be used for classification and prognosis of cancers, besides predicting the response to therapy.[139] Positron emission tomography (PET), which allows non

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invasive and quantitative analysis of various biologic processes, uses a glucose analogue(2deoxy-D-glucose) labelled with a positron emitter Fluorine 18; FDG that is partially metabolized

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and trapped as its phosphate (2-DG-6-P) in the tumour tissue thus localizing the tumour. [140] The extent of increase in glucose utilization measured by FDG-PET has been correlated with the degree of malignancy in some of the tumours.[141] Glucose utilization is also inversely correlated with treatment response in a number of tumours, while changes in tumour glucose utilization during the first weeks of chemotherapy are significantly correlated with patient outcome.[142,143] Glucose utilization appears to be a useful metabolic marker for diagnosis, prognosis and prediction of tumour response to a variety of therapies.[144]

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19. MOLECULAR MARKERS

21.1.CA15-3

CA 15-3 is one of the first circulating prognostic factors for breast cancer. Preoperative concentrations thus might be combined with existing prognostic factors for predicting outcome

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in patients with newly diagnosed breast cancer. At present, the most important clinical

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not assessable by existing clinical or radiologic procedures.[145]

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application of CA 15-3 is in monitoring therapy in patients with advanced breast cancer that is

CA15-3 (Cancer Antigen 15-3) is a tumor marker used to monitor breast cancer. It is found on

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the surface of many types of cancer cells and shed into the blood stream. It is used to monitor advanced, i.e. metastatic, cancer[146] Elevated CA15-3, in conjunction with alkaline

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phosphatase (ALP), was found to be associated with an increased chance of early recurrence in breast cancer.[147] CA15-3 and associated CA27.29 are different epitopes on the same protein

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antigen product of the breast cancer-associated MUC1 gene. CA27.29 has enhanced sensitivity and specificity and has therefore surpassed CA15-3 as a serum tumor marker. CA27.29 is

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elevated in 30% of patients with low-stage disease and 60 to 70% of patients with advancedstage breast cancer.CA15-3 protein which involves in cell protection and lubrication, is a

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member of the family of proteins known as mucins. It plays a role in reducing cell adhesion and is found throughout the body. Elevated levels of this antigen are found mainly in breast cancer

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where it appears to be involved in metastasis.[148] CA15-3 level is elevated in nearly 11 per cent of women with operable breast cancer, and 60 per cent of women with metastatic disease. Pre-

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operative concentration of CA15-3 is associated with worse prognosis than those with low concentrations.[149] CA15-3 appears to be a marker for individualizing therapy in patients with breast cancer, where patients with high CA 15-3 show good response to aggressive treatments.[150] Serum CA15-3 has been used as a surrogate marker of disease bulk to monitor metastatic breast cancer patients undergoing treatment and for the preclinical detection of tumour recurrence. Elevated levels of CA-15-3 has also been found in patients with other cancers (lung, colorectal, ovarian, pancreatic) and hepatic dysfunction. The upper limit of normal level of this marker is 25U/ml.[151]

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21.2 CA 27.29

CA 27.29 is another marker to monitor breast cancer patients. • This test measures the same marker as CA 15-3 but in a different way & does not appear to be any better in detecting early or advanced disease. • It can also be raised in other cancers and in some non-cancerous conditions.

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• Serum markers such as CA 15-3 & CA 27.29 may be used to measure results of

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cancer responds to treatment and rise if disease progresses.[151]

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treatment for patients with advanced breast cancer. Blood levels should go down if

21.4 miRNAs as Novel Drug Targets for Cancer Treatment

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The role of miRNAs in tumor initiation, progression, and metastasis in human cancers strongly suggest miRNAs as novel drug targets or therapeutic tools to develop novel strategies for the treatment of human cancers. Plausible approaches could be through either downregulating

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“oncogenic” miRNAs or upregulating “tumor suppressor” miRNAs. Two approaches have been

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tested for inhibition of the levels of miRNAs, namely, short oligonucleotides complementary to miRNAs (antagomir or antimir) [158] and miRNA sponges, which refer to synthetic mRNAs

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containing multiple binding sites for a specific miRNA and thereby competitively sequestering the endogenous miRNA [159] To achieve efficient binding with a miRNA, antagomirs and

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antimirs can be chemically modified as 2-O-methyl oligoribonucleotides and locked nucleic acid (LNA), respectively [158] elevating the level of endogenous miRNAs could be achieved by the

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delivery of synthetic miRNAs or DNA constructs that code for specific miRNAs. Efficient, safe, and tissue-specific delivery of synthetic miRNA, antagomir, or antimir has been tested in laboratories with certain degree of success , similar to other gene therapy approaches,. Delivery strategies include uses of plasmids, viral vectors or transposons, as well as cationic liposomes coupled with monoclonal antibodies to direct membrane-permeable reagentsconjugated miRNA to specific organs such as the lung and the liver. PC3 prostate cells express miR-221/222 at high levels. Treatment of mice bearing established subcutaneous PC3 tumor xenografts with anti-miR-221/222 antagomirs significantly suppressed tumor growth with a long-term effect of tumor reduction, suggesting the clinical applicability of antagomir [160]

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LNA-antimir oligonucleotides have also been tested in vitro as well as in vivo. Treatments with LNA-antimirs against miR-16, miR-21, and the miR-17-92 cluster were tested in cancer cell lines derived from glioblastoma, colon cancers, breast cancers, and lung cancers.[161] Administration of LNA-antimir to mice effectively blocks liver-specific miR-122 expression in vivo [162]. miR-127 is constitutively expressed in normal cells but epigenetically silenced in cancer cells. Treatment of cancer cells with chromatin-modifying drugs such as 5-Aza-CdR and

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PBA that inhibits DNA methylation significantly elevated the expression of miR-127 and

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inhibited cell proliferation, implicating an application of epigenetic approaches for cancer

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treatments.[163] Investigation on the roles of miRNA in cancer represents a developing and promising research field in the war against cancer. As new knowledge and technologies

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continuously emerge, novel and effective anti-cancer strategies are becoming increasingly possible. Clinical management of human cancers will greatly benefit from the development of

AN

miRNA-based diagnostic and therapeutic approaches, and the pharmaceutical industry is also welcoming a new challenging opportunity in this exciting process. On the other hand, while the

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discovery of miRNA and the identification of its roles in cancer pathogenesis provide enormous promises in improving the outcome of cancer management, the research community as well as

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the industry face challenges in developing and delivering applicable and practically effective miRNA-based anti-cancer technologies. Like all other sequence specificity-based strategies,

PT

miRNA targeting can be accompanied by unwanted non-specific actions, and the resultant offtarget effects could be even more daunting for miRNAs because miRNAs are characteristic of

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using imperfect complementarity to interact with their target sequences, displaying an intrinsic feature of the so-called multi-specificity. Furthermore, while the “multi-specific” feature enables

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a single miRNA to target more than one mRNA species, an individual mRNA can be synergistically regulated by multiple miRNA species. These features have obviously increased the complexity in developing effective miRNA-based anti-cancer approaches with minimal adverse effects. In addition, the development of new vehicle systems with low pharmacological toxicities is also urgently needed for the delivery of miRNAs or anti-miRNA inhibitors in vivo, particularly for systemic delivery. In this context, novel chemistry for miRNA modification, improved viral or non-viral vectors, and nanotechnology-based transportation tools are expected to find stages in the course of implementing safe and effective strategies for the detection and delivery of miRNAs.

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22.TREATMENT

Depending on the stage of the breast cancer treatment options vary. Current treatments for breast cancer include surgery, radiotherapy, chemotherapy, hormonal and targeted therapies. These

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therapies may be used alone or in combination depending on the stage of the disease.

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22.1 SURGERY

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For breast cancer that has not spread to other parts of the body and for more advanced stages of the disease surgery is the main treatment option. The types of breast cancer surgery differ in the

AN

amount of tissue that is removed with the tumour.[164] They are Breast conserving therapy or ‘Lumpectomy’ which involves the removal of the cancerous area, the surrounding tissue and in some cases the lymph node, whist aiming to maintain a normal breast appearance after surgery.

M

In ‘Partial Mastectomy’ or ‘Quadrantectomy’ a larger portion of tissue is removed. ‘Total

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Mastectomy’ is performed in an attempt to further cancer prevention. This surgery involves the removal of the entire breast, without the removal of lymph nodes. [165]

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22.2 RADIOTHERAPY

To reduce the chances of the cancer recurring radiation therapy in addition to surgery and

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chemotherapy can be used. It can be given after surgery (known as adjuvant treatment) or in conjunction with chemotherapy prior to surgery (neoadjuvant therapy) to shrink the tumour. In

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advanced metastatic breast cancer to help alleviate symptoms radiotherapy can also be used without surgery .

22.3 CHEMOTHERAPY Chemotherapy drugs can be given intravenously (directly into the blood), or orally in a tablet. Chemotherapy may be given prior to surgery (neo-adjuvant) with the aim of reducing tumour size and the need for extensive surgery, or after surgery (adjuvant) to reduce the chances of the cancer coming back. When the cancer has spread to other parts of the body (metastatic), chemotherapy may be used to reduce symptoms, improve quality of life and extend survival. Chemotherapy is typically associated with adverse side effects such as fatigue, nausea and

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diarrhoea; this is because of its toxic nature and non-specific mode of action, which means that all cells are attacked (even healthy cells). 22.4 HORMONAL THERAPY In the treatment of patients with Hormone Receptor-Positive breast cancer medicines that block or inhibit the actions of the hormones oestrogen and progesterone are often used. 22.5 TARGETED THERAPY

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Targeted therapy includes the use of monoclonal antibodies, vaccines and gene therapies.

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Targeted therapies are also known as biological therapies. Targeted therapies precisely target

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cancer-specific processes, making them effective and less toxic to non-cancerous, healthy cells. Several types of targeted therapy exist for the treatment of advanced breast cancer. These may be

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given after chemotherapy as maintenance or in conjunction with other therapies.[166]

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22.6 TREATMENT RESISTANCE

Mutations in MDR genes (which codes p glycoprotein) and breast cancer resistance protein

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(Bcrp) also play important roles in resistance and therapeutic outcome of breast cancer therapy. Glycoprotein non-metastatic B (GPNMB, also named as Osteoactivin) enhances breast cancer

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metastasis in an in vivo mouse model. It also has been studied as a prognostic indicator of recurrence. The data suggested this glycoprotein as a novel therapeutic target in breast cancer.

poor outcome.[167]

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GPNMB usually express in basal/triple-negative subtype of breast cancer and is associated with

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Fetuin-A is a serum component protein which forms approximately 45% of noncollagenous glycoproteins which is synthesized by the liver and excreted into plasma. It is a

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conserved member of the cysteine protease inhibitors which contains the TGF-β receptor II homology 1domain (TRH1). It is a glycoprotein which has its role in mammary tumorigenesis. It is able to compete with epithelial cells for TGF-β . The possible sequestration of TGFβ by fetuinA could affect TGFβ signaling in breast epithelial cells as previously reported for intestinal epithelial cells. Fetuin-A shows reduced incidence of mammary tumors for breast cancer by more than 60% and increases tumor onset. Another tumor-enhancer property of fetuin-A is its stabilizer effect matrix metallo proteinases in the extracellular matrix. Consequently, they can drive the “tumor islands” to invade the stroma metastasize to other organs. Stronger TGF-β signaling in the absence of fetuin-A exert suppressor effect on cell proliferation through increase

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in is ARF-p53 expression, whereas the sequestration of TGF-β by fetuin-A, results in reduction of its signaling in epithelial cells and inactivation of ARFp53 which is parallel with shortening the latency of mammary tumorigenesis and implications of breast cancer development[168] ALDH3A1 and MET were established to partially be associated with the chemo resistance role of MTDH/AEG-1 in MDA-MB-231 breast cancer cells. Some other genes also contribute

to

chemo

resistance

including

drug-metabolizing

enzymes

for

different

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chemotherapeutic agents, such as dihydropyrimidine dehydrogenase (DPYD), cytochrome

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P4502B6 (CYP2B6), dihydrodiol dehydrogenase (AKR1C2), and the ATP-binding cassette

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transporter ABCC11 for drug efflux.[169]

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23.PROGNOSIS

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23.1 STAGING AND PROGNOSIS

The prognosis for breast cancer generally depends on its stage and there are typically five stages

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(0 to 5) with sub-stages: Table 2.Represents the prognosis of breast cancer according to stages of cancer and tnm classification of cancer. Table 2.represents the prognosis of breast cancer

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according to stages of cancer and tnm classification of cancer

The effect of BMI on

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23.2 OBESITY

outcomes for defined breast cancer patient cohorts and treatment

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regimens. Retrospective analyses of 2887 node-positive breast cancer patients enrolled in the randomized phase III trial BIG 02–98 and of 1310 node-positive high-risk breast cancer patients

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enrolled in the randomized phase III ADEBAR trial showed worse DFS and OS rates for obese (BMI ≥ 30) compared with non-obese (BMI < 30) women.[170,171] Single-study analysis of 3754 patients demonstrated that only severe obesity (BMI ≥ 40) is associated with worse outcomes in operable high-risk early breast cancer, especially in the triple-negative subtype. More studies are needed to evaluate the effect of obesity on outcomes in breast cancer patients with respect to both the obesity level that constitutes an independent risk factor for reduced survival, and the effect of obesity in different tumor subtypes.[172]

24.VACCINES

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Immune recognition of cancer in the autologous host has been provided by the identification of human tumour antigens [173,174] and by the verification of cancer immune surveillance. [175]Cancer vaccines are a direct application of this knowledge and are based on the principle that a rigorous auto tumour lytic immune response can be induced in cancer patients by immunization with tumour-associated antigens. Successful development of immunotherapeutic breast cancer vaccines hinges on the identification of appropriate target antigens and the

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establishment of effective immunization strategies, as well as on our ability to devise methods to

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circumvent immune escape mechanisms utilized by the evolving tumor. Preliminary progress in

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meeting these challenges is being made, as demonstrated by the ability of cancer vaccines to induce antigen-specific T lymphocyte responses and objective clinical responses in cancer

US

patients. Although the results of recent clinical trials are promising, it should be noted that these are early-stage vaccine trials involving small populations of mostly end-stage melanoma patients,

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and are subject to variable patient and tumor responses.

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24.1 TARGET MOLECULES FOR ANTIGEN-SPECIFIC BREAST CANCER VACCINES Both immunogenicity in cancer patients and restricted tissue expression are characteristics used

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to define antigenic targets for cancer vaccines. Immunological methods of gene discovery, such as CD8+ and CD4+ T cell epitope cloning[173,176] and serum antibody expression cloning

PT

(SEREX) (WHO Cancer factsheet 2011), have led to the identification of tissue-restricted tumor antigens that are recognized by the immune systems of cancer patients and have added to the list

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of target antigens applicable to breast cancer . These antigens fall into several categories, such as differentiation antigens, cancer-testis antigens, amplified/overexpressed gene products, and

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mutational antigens. One of the first target molecules to be examined in the context of a breast cancer vaccine is carcinoembrionic antigen (CEA), a differentiation antigen of the gut, expressed exclusively in normal colonic epithelium and approximately 50% of breast cancers.[177] With regard to clinical trials, Morse and colleagues have observed objective responses in patients with metastatic disease, including breast cancer, following immunization with dendritic cells pulsed with an human leukocyte antigen (HLA)-A2 restricted peptide of CEA [178] Recently, a new differentiation antigen of the breast, NY-BR-1, was identified by SEREX analysis and was found to be expressed exclusively in normal testis and breast, as well as in 80% of breast cancers . NYBR-1 is recognized by high titered serum IgG antibodies present in breast cancer patients, and its

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ability to induce a cellular immune response is under investigation. Potential targets for antigenspecific

breast

cancer

vaccines

and

their

frequency

of

mRNA

expression,

amplification/overexpression1 or mutation2 in breast cancer Cancer-testis antigens represent a group of immunogenic proteins expressed exclusively in normal germ cells of the testis and embryonic ovary, and a percentage of various cancers. The melanoma antigens MAGE, BAGE, and GAGE are prototype cancer-testis antigens, first

T

identified by cloning epitopes recognized by CD8+ T lymphocytes of melanoma

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patient.[173,179] SEREX analysis has also led to the identification of cancer testis antigens,

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including New York Esophageal-1 (NY-ESO-1) , cancer testis-7 (CT-7), and the synovial sarcoma-x (SSX) family of antigens.[176] The enormous potential of CT antigens as vaccine

US

targets is based on their restricted expression pattern and their high frequency of immunogenicity in cancer patients. Results of recent clinical trials using NY-ESO-1[180] and MAGE-3 [181] as

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target antigens have been promising in terms of inducing antigen-specific T cells in vivo and, in some cases, concomitant disease regressions.

M

Mutated and amplified gene products represent another group of target antigens. The Her2/neu oncogene is amplified in approximately 40% of breast cancers, and Her-2/neu-specific T

ED

cell responses have been observed in patients vaccinated with major histocompatibility (MHC) class II binding peptides derived from Her-2/neu [182] The p53 tumor suppressor gene is

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frequently mutated in breast cancer and is associated with an autologous antibody response in breast cancer patients [183] The large number of different p53 mutations makes targeting

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mutated p53 epitopes impractical. On the other hand, mutations increase the cellular half-life of p53, causing it to be overex-pressed in cancer, indicating that immunization with wild type p53

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may be an alternative. In fact, cytotoxic T lymphocyte (CTL) clones reactive against wild type p53 were generated from precursors present in the peripheral blood lymphocytes of healthy individuals, and were capable of lyzing several human tumor cell lines. [184] Three additional antigens recognized by the humoral immune system of breast cancer patients, NY-BR-62, NYBR-85, and tumor protein D52, were found to be overexpressed in 60%, 90%, and 60% of breast cancers, respectively.Their significance in relation to breast cancer vaccines is being investigated. [185]

25.CONCLUSION

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The article gives a clear view on the molecular pathology, the diagnostic options and the updated treatment options of breast cancer.It has been known that genes are the key players in the basic pathogenesis and the diagnostic part of breast cancer. Although Metabolism of cells plays a basic role in the progression of breast cancer and thier metastasis, this article mainly highlights the diagnostic role of metabolic markers in breast cancer. Hence the treatment modalities can be

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planned at the genetic level at the metabolic level or at the molecular level also.

Garcia M et al. Global Cancer Facts & Figures. Atlanta, GA: American Cancer Society, 2007. WHOCancer

fact

sheet

updated

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2

US

1

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Abbreviation

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ER - estrogen receptor PR -progesterone receptor PCB's - Organochlorines include polychlorinated biphenyls BRCA-1 and BRCA-2 - breast-cancer susceptibility gene 1 and 2 SCD - serine cluster domain

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1. 2. 3. 4. 5.

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Figure.1 SHOWS GENETIC FACTORS INVOLVED IN THE BREAST CANCER.[24,25]

Figure.2 SHOWS THAT MTDH/AEG-1 PROMOTES TUMOR PROGRESSION THROUGH THE INTEGRATION OF MULTIPLE SIGNALING PATHWAYS.[103]

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Figure.3 SHOWS THE ROLE OF APC IN BREAST CANCER PROGRESSIONS[104]

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FIGURE. 4. ILLUSTRATES THE PATHWAY OF CARCINOGENESIS AND TUMOUR MARKERS INVOLVED IN THE BREAST CANCER METASTASIS[120-122]

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TABLE: 1 REPRESENTS THE RISK FACTORS FOR BREAST CANCER PROGRESSIONS.[17]

Elderly Developed country Age at menarche before 11years

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Age at menopause after 54 years

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Age at first full pregnancy in early 40s

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Family history Previous benign disease (atypical hyperplasia)

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Cancer in the other breast Diet with high intake of saturated fat

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ACCEPTED MANUSCRIPT 56 TABLE 2.REPRESENTS THE PROGNOSIS OF BREAST CANCER ACCORDING TO STAGES OF CANCER AND TNM CLASSIFICATION OF CANCER

Stage

TNM

5-year

Tis N0 M0

99%

I

T1 N0 M0

92%

IIA

T0

N1

M0 82%

T1

N1

M0

CR

T2 N0 M0 T2

N1

T3 N0 M0 T0

N2

T1

N2

M0

T2

N2

M0

T4

Any

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IIIB

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T3 N1, N2 M0

M0 47%

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IIIA

M0 65%

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Survival

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M0 44%

Any T N3 M0 Any T Any N M1

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T= Status of Primary tumor; N = Regional lymph nodes; M = Distant Metastases

ACCEPTED MANUSCRIPT 57 Research Highlights

Breast cancer is more common among urban females. It has been estimated that in India there will be 1,55, 000 new cases of breast cancer and 76,000 deaths due to breast cancer in 2015. Many factors play a key role in the progression of breast cancer but there is no evidence that proves or cure its progressions. Therefore, It is very important to take necessary early steps to screen , diagnose and treat the breast cancer as early as possible to save millions of lives. The present review high lights the significance of so many factors responsible for the progression

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of this disease, which includes pathological causes, roles of various molecular and biochemical markers, genetic

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and also emerging treatment modalities along with the existing ones like vaccines.