Molecular Diagnosis of Breast Cancer

Abstract

Breast cancer is the most commonly diagnosed cancer in the world. Clinical manifestation and instrumental methods like mammography, ultrasound, and MRI are widely used in detecting breast cancer, however, molecular diagnosis is an indispensable aspect of breast cancer diagnosis processes. The method is a cornerstone for assessing the risk factors, screening the potential case, diagnosing the disease accurately, selecting the proper treatment procedures, prescribing the most effective drug, and monitoring the treated patients. Along with it, the chapter has also covered the different methods of molecular genetic diagnostic procedures used in clinical practice and for research, with their advantages and shortcomings. Modern technologies have allowed oncologists to dive deeper into the different types of breast cancer in order to find the most effective treatment for the patient, leading to the era of precision medicine in the field of oncology.

1. Introduction

Breast cancer is a form of malignant tumour in which breast tissue grows uncontrollably. It has been enlisted to be the most commonly diagnosed cancer in the world. According to the data, around 2.26 million cases of breast cancer were recorded in 2020, followed by lung cancer (2.21 million) and prostate cancer (1.41 million) [1]. In order to identify the cancer in its early stage, screening is done routinely. The screening usually consists of instrumental methods of diagnosis like mammography and ultrasound. When there is a suspicion of cancer, the patients undergo a biopsy and various molecular studies are done to diagnose the disease, identify its molecular subtype, and select the most appropriate treatment. However new genetic tools have recently been discovered that aid in screening and diagnosing breast cancer as well. Finally, after treatment has been completed, certain biomarkers are monitored to observe the progression/stabilization of the disease.

Currently, molecular biological technologies are used to solve the whole range of tasks related to diagnosis, therapy planning, monitoring, and prognosis in breast cancer.

But, first of all, we need to understand what causes breast cancer. The most widely accepted theory of its origin is related to genetics, however exact aetiology of cancer is still shrouded in mystery. Nevertheless, there are some factors that seem to enhance the growth and development of breast cancer. For studying breast cancer, we first need to understand what those risk factors are, which are illustrated in Table 1.

Risk factor	Remarks
Sex	Women are more common victims of breast cancer than men.
Age	It is the second most important risk factor for breast cancer. The incidence of breast cancer is found to be increased with increasing age. According to an article in 2017, the risk of breast cancer is directly proportional to age [2]: From birth till age 49: 1 in 52; 50–59: 1 in 44; 60–69: 1 in 29; >70: 1 in 15
Family history	Nearly 25% of breast cancers have been found to be associated with family history. In one of the UK cohort researches, individuals with one-first degree relative suffering from breast cancer has an increased risk of breast cancer by a factor of 1.75. The factor increases to 2.5 if the individual has two or more first-degree relatives with breast cancer [3]. This is partially found to be related to BRCA1 and BRCA2 genes.
Reproductive factors	The number of ovulations is directly proportional to the increased risk of developing breast cancer. Hence, reproductive characteristics like early menarche, late menopause, late age of first pregnancy, and low parity increase the risk of developing breast cancer. Breast cancer risk is increased by 3% for each 1-year delay in menopause. Each additional birth or each 1-year delay in menarche reduces the risk of breast cancer by 5% or 10%, respectively [4, 5].
Estrogen	Estrogen levels are associated with the risk of breast cancer. Estrogen is endogenously produced by the ovary in premenopausal women and exogenously received in the form of oral contraceptives or hormone replacement therapy, which may trigger breast cancer [6]. Even though formulations in oral contraceptives have been upgraded to reduce side effects, the risk was still found to be higher than 1.5 for African-American women and Iranian populations [7, 8].
Lifestyle	Breast cancer risk can be increased by contemporary lifestyle factors such as excessive alcohol use and dietary fat consumption. Alcohol drinking can stimulate the estrogen receptor pathways and raise blood levels of hormones associated with estrogen. Consumption of 35 to 44 grams of alcohol per day can raise the risk of breast cancer by 32% [9, 10].; Mutagens from cigarette smoke have been found in the breast fluid of non-lactating women, despite the fact that the link between smoking and the risk of breast cancer is still debatable. Women who smoke and drink also have a higher chance of developing breast cancer [11]. As of today, mounting data show that smoking, especially when starting young, increases the chance of developing breast cancer [12, 13].
Racial disparity	Breast cancer varies in different races. The frequency of having breast cancer follows the order: White women>Black>Asian/Pacific>Islander>Hispanic>American Indian/Alaska Native. On the other hand, the fatality of breast cancer amongst the different ethnic groups has the order: Black women>White>Hispanic>Asian/Pacific>Islander>American>Indian/Alaska Native women [14].

Table 1.

Risk factors of breast cancer.

As there are many factors that need to be accounted for determining the risk of having breast cancer, there exists a number of online calculators that compute the risk of having cancer, based on the presence/absence of these factors. Some of the calculators are as follows:

• https://bcrisktool.cancer.gov/calculator.html

• https://ibis.ikonopedia.com/

• https://www.bu.edu/slone/bwhs-brcarisk-calculator/

Besides environmental factors, there are genetic factors as well which influence breast cancer. There are several genes that involve in breast cancer, however, the gene mutations that frequently cause breast cancers are BRCA1, BRCA2, PTEN, TP53, CDH1, STK11, CHEK2, BRIP1, ATM, and PALB2 [15]. These gene mutations can have high risk or low risk (Table 2). If the gene mutation occurs in the gene of high risk, there is a greater probability of having second breast cancer, so the patients may be given the option to undergo prophylactic bilateral mastectomy.

Risk	Gene	Known/possible function
High risk	BRCA1 (BReast CAncer1)	BRCA1 is a tumour suppressor gene. It is found to regulate the cell cycle, maintain genome stability, and repair DNA damage [16]. Patients with these mutations are recommended to undergo bilateral salpingo-oophorectomy for reducing the risk of another breast cancer [17].
	BRCA2 (BReast CAncer2)	BRCA2 is also a tumour suppressor gene that regulates the mechanism of DNA repair and cell death pathway and thus controls uncontrolled cell division [18]. Patients with these mutations are also recommended to undergo bilateral salpingo-oophorectomy for reducing the risk of breast cancer [17].
	ATM (Ataxia Telangiectasia Mutated)	Whenever there is a DNA double-strand breakdown, ATM gets activated, which in turn phosphorylates many downstream tumour suppressor genes such as BRCA1, P53, CHK1, and CHK2 to prevent uncontrolled cell division [19].
	PALB2 (partner and localizer of BRCA2)	PALB2 functions as a tumour suppressor gene and participates in the maintenance of genome integrity [20].
	PTEN (Phosphatase and tensin homolog)	PTEN is a phosphatase and is responsible for the regulation of cell signalling pathways like PI3K/Akt/mTOR, FAK/p130cas, and ERK/MAPK [21, 22, 23, 24]. It plays a key role in many cellular functions like inhibition of cell adhesion and migration, apoptosis, blockade of the cell cycle and cell proliferation, inhibition of angioneogenesis, DNA repair, and metabolism [22, 23, 25, 26, 27].
	TP53 (tumour protein p53)	TP53 is a tumour suppressor gene that codes for a protein that regulates cell cycle arrest, cellular senescence, apoptosis, metabolism, DNA repair, and other processes following cellular stress [28].
	STK11/LKB1 (serine-threonine kinase 11/ liver kinase B1)	The STK11/ LKB1 is a gene that encodes for a tumour-suppressing enzyme called serine/threonine kinase 11, thereby controlling the uncontrolled growth and division of cells [29].
Low risk	CDH1 (Cadherin 1)	The CDH1 gene is responsible for encoding the E-cadherin protein, which is key for maintaining pluripotency and self-renewal of embryonic stem cells and neural stem cells [30, 31, 32]. Dysfunction of CDH1 is thought to increase the risk of development of malignant tumour and metastasis [33].
	CHEK2 (Checkpoint kinase 2)	When DNA is damaged, CHEK2 is activated which plays an important role in pathways that govern DNA repair, cell cycle arrest, or apoptosis [34].
	BRIP1 (BRCA1 Interacting Protein 1)	BRIP1 is a tumour suppressor gene that repairs DNA damage and preserves genetic stability [35].

Table 2.

Genetic factors of breast cancer.

The presence of a mutation, under certain circumstances, can lead to the development of a malignant phenotype (the appearance of a tumour).

The ultimate change, that converts normal functioning cells into cancerous cells is due to a change in its phenotype. A phenotype is a combination of observable characteristics or traits in an organism. The characteristics might be its physical form, chemical properties, or physiological behaviour which is determined by the interaction between its genetic makeup and surroundings. In a nutshell, it can be said that a phenotype is the set of observable characteristics of an individual resulting from the interaction of its genotype with the environment. There are few changes in the phenotypes which occur in cancerous cells. They are as follows:

• self-sufficiency in growth signals

• insensitivity to anti-growth signals

• apoptosis evasion

• limitless replication potential

• sustained angiogenesis

• tissue invasion and metastasis

• abnormal metabolic pathways

• immune system evasion

Such phenotypic modification may be expressed due to changes in genotype. However, the change may occur in different levels (Table 3).

Level	Mechanism	Detection methods	Example
Genetic level	Chromosomal translocations point mutations Amplification Genetic instability	Site-specific Polymer Chain Reaction (PCR), methylation-specific PCR, Single-Strand Conformational Polymorphism (SSCP), heteroduplex analysis, Hybridization, Fluorescence In Situ Hybridization (FISH), Sequencing.	P53, RAS, ER, RAF B, BRCA1, BRCA2, P16 MUC-1, integrin, ERBB-2
Epigenetic level	DNA methylation Histone acetylation Chromatin modifications RNA-mediated gene silencing		P16, GSTP1, APC, LINE, cellular Myc
RNA	Alternative splicing Life span change Epigenetic changes	Quantitative PCR, Serial Analysis of Gene Expression (SAGE) hybridization, differential display PCR (DD-PCR)	PSA, MUC-18, AFP.
Protein	Post-translational modification Concentrations	Use of antibodies (Enzyme-Linked Immunosorbent Assay (ELISA), immunohistochemistry, Western blot), Protein Truncation Test (PTT), Gas Chromatography–Mass Spectrometry (GC-MS)	A panel of trefoil factor (TFF) 1, TFF2, and TFF3
Low molecular weight metabolites	Profiles of low molecular weight metabolites	GC-MS, ELISA, Radioimmunoassay (RIA)	elevated choline, low glycerophosphocholine, and low glucose

Table 3.

Different levels of change which are finally manifested into phenotype.

If the patients have been genetically predisposed to mutation of genes that increase the risk of breast cancer, such breast cancers are called hereditary cancer. On the other hand, it should be emphasized that a malignant tumour can also occur in the absence of hereditary mutations. Changes can occur already in the somatic cells of the body as well. In such cases, the breast cancer would be sporadic. The clinical properties and treatment approaches for these two variants of breast cancer differ.

2. Screening

The effectiveness of breast cancer therapy highly depends on the stage at which the cancer is detected. Therefore, methods and approaches for early detection of breast cancer are extremely important.

The first clinical sign of breast cancer starts with patients complaining about a lump in the breast. Around four-fifths of the patients have this symptom. Besides it, some may suffer from breast pain, and abnormalities of nipple, and breast skin [36]. The next step for those patients is to undergo instrumental methods of diagnosis, i.e. mammography (for females >40 years old), ultrasound (for females <40 years old), and thermography. When the result is not decisive, an MRI can also be taken. These days, artificial intelligence has also been accompanied with these instrumental methods of diagnosis to aid in making correct predictions. Along with the instrumental methods, recent studies have come up with new contemporary approaches that can also be utilized in diagnostic procedures. Some of the oncomarkers that have been actively studied and can be used in diagnostic procedures are given in Table 4.

Oncomarker	Remarks	Sensitivity (%)	Specificity (%)
Circulating tumor DNA (ctDNA)	Tumours can release their DNA fragments into the bloodstream which are called ctDNA. It has only been used in research and clinical trials, but it may be used in usual clinical practice as well, once the methods are standardized. It can be implemented in screening, diagnosis, follow-up, treatment, and in metastatic breast cancer [37].	88 [38]	98 [38]
Micro-RNA	Micro-RNAs are non-coding molecules but by complementary binding, they can regulate the genetic activity of the cell. There are many RNAs which act as biomarkers for breast cancer diagnosis [39]. Specific micro-RNAs have been found to prognosticate the effectiveness of certain drug treatments as well [40]. Although the preliminary results are promising, large-scale experiments need to be conducted to validate the result so that it could be used in clinical practice.	96.95 [41]	100 [41]
linear non-coding single-stranded RNA	They are the short-length RNA molecules that are involved in transcription and translation. Different types of these RNAs can provide information about properties of the breast cancer, metastasis, and resistance to chemotherapy [42]. A clinical trial is being conducted to assess the efficacy of these RNAs [43].	92 [42]	74 [42]
Circular RNA	They are covalently bound circular loops of RNA. They can regulate gene expression via micro-RNA sponging. Some of the circular RNAs have been found to have diagnostic and prognostic roles in breast cancer [44, 45]. However, the use of circular RNA in breast cancer is still in its infancy and exploratory experiments have to be carried out to elucidate its capabilities [46].	77 [47]	71 [47]
Blood-based DNA methylation	Methylation changes in DNA in the blood samples of peripheral blood have been associated with breast cancer [48]. Increased DNA methylation within functional promoters across the genome and decreased DNA methylation in other regions is the hallmark of breast cancer [49]. However, the evidence is still limited for blood-based methylation markers to be used in the general population for early detection of breast cancer. So, a large and methodologically rigorous epidemiological experiment has to be conducted to validate the use of blood-based DNA methylation in detecting breast cancer [50].	88.9 [51]	80.6 [51]
Autoantibodies	As the tumour cells are genetically unstable, the proteomes of the tumour are modified by phosphorylation, acetylation, and glycosylation, forming tumour-associated antigens. As a result, the body makes autoantibodies against those tumour-associated antigens. Detecting those autoantibodies aids in the diagnosis of breast cancer far before symptoms are clinically manifested [52, 53]. However, the autoantibodies are not used in clinical practice because their sensitivity and specificity are not higher than the methods that are currently in use (mammography). Furthermore, there exists no standard for detecting these autoantibodies in breast cancer detection [52].	24 [54]	96 [54]

Table 4.

Some of the diagnostic oncomarkers.

After the suspicious lesion has been identified in the breast tissue, the next step is to undergo a biopsy to distinguish it from other benign conditions and to study the morphological features of the tumour for determining the prognosis and selecting the optimal therapy. The suspected lesion in the breast can arise usually from two different tissues—ducts (80% of cases) or lobules (10% of cases) of the glandular breast tissue. The less common 10% of breast cancer consists of mucinous, cribriform, micropapillary, papillary, tubular, medullary, metaplastic, and inflammatory carcinomas [55, 56]. On the other hand, breast cancer can also be non-invasive (stage 0) or invasive (stages I to IV). Depending upon its origin and invasiveness, breast cancer can be classified as:

• Ductal carcinoma in situ (DCIS)

• Invasive ductal carcinoma (IDC)

• Lobular carcinoma in situ (LCIS)

• Invasive lobular carcinoma (ILC)

IDC is the most common form of breast cancer, accounting for around 55% of all breast cancers [57].

After the biopsy of the lesion is taken, the material is thoroughly studied and the phenotype is revealed through immunohistochemistry (IHC). On the basis of IHC, breast cancer has four primary molecular phenotypes, defined in large part by hormone receptors and other types of proteins involved (or not involved) in each cancer (Table 5) [61].

Molecular biological subtype		Clinic-pathological subtype	% of relapse [58]	Frequency of metastatic sites [59]	Treatment [60]
Luminal A		ER+ HER2- Ki67 < 20% PR > 20%	5.02	Multiple site-50.9%; One site-49.1%; Bone metastasis-30%; Lung metastasis-5%; Liver metastasis-3.5%; Others-10.6%	Mostly hormonal therapy
Luminal B	Luminal B HER-	ER+ HER2- Either Ki67 > 30% or PR < 20%	7.88		Hormonal therapy + chemotherapy (docetaxel + cyclophosphamide or doxorubicin + cyclophosphamide + docetaxel / Paclitaxel)
	Luminal B HER+	ER+ HER2+	6.61	Multiple site-58.8%; One site-41.2%; Bone metastasis-22.9%; Lung metastasis-1.5%; Liver metastasis-6.1%; Others-10.7%	Hormonal therapy + chemotherapy (docetaxel + cyclophosphamide or doxorubicin + cyclophosphamide + docetaxel / Paclitaxel) + anti-HER2 therapy (trastuzumab)
HER+		ER- HER2+	13.1	Multiple site-50%; One site-50%; Bone metastasis-16.2%; Lung metastasis-5.4%; Liver metastasis-13.5%; Others-14.9%	Chemotherapy + anti-HER2 therapy (docetaxel + cyclophosphamide or doxorubicin + cyclophosphamide + docetaxel / Paclitaxel)
Basal type		ER- PR- HER2-	16.76	Multiple site-53.6%; One site-46.4%; Bone metastasis-16.8%; Lung metastasis-9.6%; Liver metastasis-6.4%; Others-13.6%	Table 6

Table 5.

The molecular biological subtype of breast cancer.

2.1 Luminal A breast cancer

Luminal A tumours are the most common molecular type of breast cancer. This biological subtype has a receptor for estrogen and/or progesterone and can be treated using drugs blocking these hormones. This form of cancer highly expresses genes responsible for a molecular cascade of estrogen gene- ESR1, XBP1, FOXA1, GATA3, TTF3, LIV3, HER4, PIK3RI as well as highly express luminal cytokeratin due to methylation of RASSF1, GSTP1, MMP7, PEG10, APC genes.

2.2 Luminal B breast cancer

These types of cancer are hormone positives as luminal A but differ by the high level of Ki67%. This is the reason why luminal B breast cancers grow faster than luminal type A. Luminal type B can be HER2 positive or HER2 negative. In the phenotype, total methylation of the genome is found, especially of genes RASSF1, GSTP1, CHI3L2.

2.3 HER2-enriched

One in five invasive breast cancers is HER2-positive, making this one of the more common breast cancer subtypes in the world. HER2-positive cancers are ER- and PR-negative and HER2-positive. GRB7, HRAS, MEK1/MEK2, AKT1 are also overexpressed in this phenotype of breast cancer.

HER2-positive breast cancer cells carry too many copies of the HER2 gene, which makes HER2-protein receptors, found on breast cells. The main function of HER2 receptor is to control how breast cells grow. When there is excess HER2, the cells absorb excess human epidermal growth factor, which makes them proliferate rapidly.

2.4 Triple-negative

In this type of cancer, the cells do not contain receptors for estrogen, progesterone, or HER2 but are characterized by high expression of oncogenes like NRAS, KRAS, C-KIT, cadherin P (CDH3), lamini alpha/gamma (LAMA5, LAMC1), MCM3/4/7, and basal cytokerain KRT5/6/17. This type of breast cancer is found in breast ducts and is very invasive in nature. The triple-negative breast cancer can be categorized into six subtypes based on their gene expression portfolio (Table 6). The subtypes are immunomodulatory (IM), luminal androgen receptor (LAR), basal-like 1 (BL-1), basal-like 2 (BL-2), mesenchymal (M), and mesenchymal stem-like (MSL) [62].

TNBC type	Characteristics	Treatment options
BL-1	DNA damage response pathway	PARP inhibitors; Platinum compounds
BL-2	Growth factor signalling, glycolysis, and gluconeogenesis	Growth signalling inhibition
LAR	High expression of genes related to hormone	Androgen receptor antagonists
M	Cell differentiation pathway, the interaction between extracellular receptors, mobility of cell	Wnt/β-catenin inhibitors; PI3K/mTOR inhibitors; TGF-β receptor kinase inhibitors
MSL	Similar to M subtype but is claudin-low and high expression of mesenchymal stem cells	PI3K inhibitors; mTOR inhibitors
IM	Immune cell process	Immune checkpoint Inhibitors

Table 6.

Characteristics and possible treatment options based on TNBC molecular subtypes.

However, recent studies using DNA microarray have identified several molecular subtypes of breast cancer—luminal A, luminal B, HER2-enriched, basal-like, claudin-low, and normal-like [63, 64, 65, 66, 67]. The classification based on complex patterns of gene expression bridges the gap between cancer behaviour and its molecular biological subtype because each breast cancer is a distinct entity based on genomic, transcriptomic, and proteomic data [68]. As each breast cancer is unique, this ushers oncology to a new era of personalized medicine, where each breast cancer can be treated individually, custom-tailored to the specific patient (Table 7).

Molecular class	Chemotherapy response	Therapy
Luminal A	Low	Tamoxifen, Fulvestrant, Aromatase inhibitors
Luminal B	Intermediate
HER2-positive	High	HER2 and kinase inhibitor: lapatinib, pertuzumab, trastuzumab, and adotratuzomab emtansine, immune cell activation (Ertumaxomab)
Basal-like	High	PARP1 inhibitor (Olaparib and Iniparib), cisplatin
Claudin-low	Intermediate/low	Inhibitor of PIK3CG combined with paclitaxel [70]
Normal-like	N/A	N/A

Table 7.

Breast cancer classification based on molecular profiling and corresponding treatment options [69].

It is to be noted that there is a disparity in molecular phenotype determined by IHC and methods of analysis of genetic expression. For example, some authors claim the disparity to be 31–59% in HER2 expression when analyzed by ICH and in situ hybridization [71, 72]. In the present day, there are several genetic tests for phenotyping and prognosticating the disease. Some of them are illustrated in Table 8.

Platform	Method	Number of genes	Indication	Prediction
Mammaprint	Chromosomal microarray, Real-time PCR	70	I and II stage, size = 5 см, estrogen (+), l/n (−)/[1−3 l/n (+)]	Prognosis of breast cancer
OncotypeDx	Real-time PCR	21	estrogen (+), l/n (−)	Prognosis of breast cancer, effectiveness of adjuvant chemotherapy
MapQuantDx	Chromosomal microarray, Real-time PCR	97/9	estrogen (+), G2	Determine grade (G) of breast cancer, the effectiveness of hormonal therapy
Breast Cancer Index	Chromosomal microarray, Real-time PCR	7	estrogen (+)	Prognosis of breast cancer, effectiveness of hormonal therapy
Oncotype Dx	Real-time PCR	21	I and II stage, estrogen (+), HER2 (−)	Effectiveness of chemotherapy, the possibility of relapse

Table 8.

Genetic tests for phenotyping and prognosticating breast cancer.

l/n = lymphnode.

Although the molecular genetic classification of the breast cancer subtype has clear benefits over the IHC subtype, molecular classification based on genetic analysis has not been implemented in routine clinical practice because of political and economic obstacles. Furthermore, IHC is readily available in every health centre, whereas it is challenging to perform molecular genetic tests due to a lack of proper equipment and expertise [73].

Finally, after selecting the adequate treatment scheme corresponding to its molecular subtype, the patient has to be monitored. For monitoring purposes, the following oncomarkers have been purposed (Table 9). Despite the existence of several oncomarkers, CA153, CA 27.29, and CEA are only used routinely in clinical practice. Other oncomarkers are specific to cancers of other organs, but they have also been investigated in breast cancer patients.

Oncomarker	Remarks	Normal value	Sensitivity (%)	Specificity (%)
CA 15-3	CA 15-3 is a carbohydrate-containing protein antigen called mucin (MUC). Its serum concentration is useful in predicting the progression of breast cancer and monitoring the efficacy of the therapy as its quantity is directly proportional to the size of the tumour and its severity (stage). It is generally recommended to monitor CA125 and CEA once every 3 months [74] as they are considered complementary [75].	≤ 30 U/mL [75]	70 [76]	96 [76]
CEA	CEA is a glycoprotein which is responsible for cell adhesion. CEA is normally produced in the gastrointestinal tissue during foetal development, but its production stops before birth. In breast cancer, CEA is elevated when there is a metastasis of the primary lesion of breast cancer. Preoperative CEA measurements have shown that its level is directly proportional to the pathological stage and tumour size and the size of a metastatic lesion. It is recommended to monitor CEA level every 2–3 months [77].	2.5 μg/L [75]	88.3 [78]	46.2 [78]
CA 27.29	CA27.29 is a carbohydrate-containing protein antigen that is also produced by MUC-1 gene as CA153. Since CA 153 and CA 27.29 are derivatives of MUC1, their clinical significance in breast cancers is comparable. However, evidence shows that CA 27.29 has high sensitivity than CA 15-3, but lacks specificity. The oncomarker is more useful in detecting the disease progression and metastatic involvement. The average time interval to evaluate CA27.29 is 5 months [79].	38 U/mL [80]	62 [81]	83 [81]
HER-2	HER-2 is a protein that is encoded by the gene erythroblastic oncogene B (ERBB2), a gene that is originally isolated from the avian genome. The protein; consists of three different parts: an extracellular ligand-binding domain E single transmembrane domain an intracellular tyrosine kinase. The extracellular domain can undergo proteolytic cleavage, thereby releasing a detectable amount of it into the bloodstream. HER-2 receptor protein has been found useful for early diagnosis of relapses and to predict the fate of metastases of breast cancer. In some studies, HER2-ECD was recommended to measure once every 4 weeks [82].	7.7 ng/ml [83]	76.92 [84]	72.92 [84]
plasminogen-activating proteins	urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR) are plasminogen-activating proteins. Their high levels usually imply a poor prognosis of the cancer. The main function of uPA is to convert plasminogen into active plasmin. It has the ability to stimulate angiogenesis, mitogenesis, cell migration and to modulate cell adhesion and to prevent apoptosis. On the other hand, PAI-1 can inhibit uPA, which is supposed to prevent metastasis. Tumour expression of PAI-1 and uPAR represent important breast cancer prognostic factors.	2.52 ng/ml [85]	44.8 [85]	85.3 [85]
Nestin	Nestin is actually a marker of neural progenitors that usually exist in stem cells of the central nervous system. However, it has also been identified in the mammary glands, especially in the basal and myoepithelial layers. They produce nestin, but the cancerous tissue produces even more nestin, which makes it an excellent diagnostic tool for breast cancer.	39.9 pg./mL [86]	84.8 [86]	65.1 [86]
HE4	HE4 is a protein that human epididymis produces. Although its secretion is related to the epididymis, researchers have found some of its diagnostic value for breast cancer. Its serum concentration has been found to increase in patients with breast cancer in comparison to healthy controls [87].	54.5 pmol/l [87]	73.3 [87]	65.3 [87]
TPA	TPA is a complex of polypeptide filaments of the cytokeratins 8, 18, and 19. Although TPA is mainly helpful in monitoring and evaluating the treatment process, it has also been found to prognosticate tumour progression and metastasis in the case of breast cancer, especially in association with other tumour markers like CEA [88].	120 U/I [89]	67.5 [89]	81.9 [89]
AFP	AFP is a protein made by growing and dividing liver cells. AFP test usually detests liver cancer and germ cell cancers, however, when combined with other tumour markers, it can also be valuable in detecting breast cancer. In terms of mechanism, AFP is thought to be involved in the regulation of cell growth and differentiation. It has been suggested that AFP may play a role in the development of breast cancer by promoting cell proliferation and inhibiting apoptosis [90, 91].	10–150 ng/ml [92]	22.2 [93]	94.33 [93]
CA199	CA199 is a protein found on the surface of certain cancer cells but it may be found in the bloodstream when shed by cancerous tissue. Usually, it is detected in pancreatic cancer, however, some studies have detected serum level changes of tumour marker CA199 in breast cancer as well [94]. It is not generally used in clinical practice due to its limited predictive value. However, an increase of CA199 above 1000 U/ml accurately predicts metastatic cancer [79].	<37 U/ml [95]	19.36 [93]	94.54 [93]
CA125	CA125 is a tumour marker that is most commonly associated with ovarian cancer, but can also be a sign of breast cancer. It has been found that about 84% of metastatic breast cancers have elevated levels of CA125 [96]. However, CA125 is not a specific marker for breast cancer and can also be influenced by benign conditions or other malignancies [97]. It is recommended to monitor CA125 every 3 months [79].	35 IU/mL [98]	93.8 [99]	28.7 [99]

Table 9.

Some of the oncomarkers for monitoring breast cancer.

3. Conclusion

Molecular diagnostic tools are involved in each and every aspect of breast cancer, starting from the screening process to monitoring the patients after treatment. But, instrumental diagnostic measures account for the majority of screening processes. If there is a suspicion, a biopsy is taken to establish a diagnosis. In order to select proper treatment, IHC is done to classify breast cancer, but it is to be noted that molecular genetic classification is more precise than IHC, however, IHC is widely used in clinical practice because it is readily available. After the patient has been treated, different oncomarkers can be checked to monitor the possible progression of the disease.

Although it has been found that contemporary treatment of breast cancer has shown better results in a considerable proportion of breast cancer patients, a certain number of patients have shown only modest benefits. Hence, the diagnostic, classification, and treatment processes have to be optimized based on novel molecular genetic parameters, which would lead to even better clinical outcomes. This would also substantially contribute to personalized medicine and result in a more effective and prolonged response, finally improving the survival rate of patients suffering from breast cancer.