Establishment And Characterization of Primary Cell Culture

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Cell multiplication which is under accurate regulation and tends to react to specific needs is faster than the death of cells. The birth and death processes of cells are in balance so that they can establish a regular condition.

Nevertheless, it sometimes happens that the controls which are the regulators of cell multiplication stop working and as a result cells start to divide and spread irregularly. In a situation where a cell like this that has descendents hereditarily prone to replicate has no response to regulation, there is a possibility for the clone of cells to develop implicitly. The final result of such a clone of unwanted cells is the formation of a mass which is commonly called a tumor. There are some tumors that do not give rise to critical health problems; however, the cause of diseases can be generally attributed to the clones of cells which spread throughout the body. Mutation brings about cancer; nevertheless, there are two differences between genetic diseases and cancer. The first difference lies in the fact that mutations in somatic cells mainly account for cancer, whereas in other genetic diseases, it is mutations in the germ line that is the detrimental factor. Some people have genetic mutations by birth and this causes them to be vulnerable to some specific types of cancer. The second difference is that a single cancer is not brought about by a single mutation; in fact, it is the result of the accumulation of about at least three and at most twenty mutations based on the type of the cancer in genes that are usually responsible for the regulation of cell duplication. Since it takes a long period of time for many mutations to accumulate, cancer is chiefly a matter of old age. (1).

The monoclonality of tumors clearly reveals that malignant growth is due to single genetic events and mutations or it is because of a mutation which leads to a steady deviation of a cell from regular program at the time of its existence and development. Mutations do not follow a fixed pattern of appearance and they do not even have any regular pathways to determine their physical nature and essences. As a result, there is no a precancer; however, according to the pathomorphology of tumors, there is vivid evidence of precancers (2).

Weinberg (3) has identified three paths for the appearance of precancer which are as follows: The first path is the emergence and replication of precursor cell of some tumors. Another type of path which was detected is that genetics changes begin to build up the chance of generation of tumor clones. The final path is to enable a non-tumor tissue, so called stroma, to make extracellular matrix, growth factors and also the factors which improve vascularisation of tumors.

1.1.1 Activation of precursor cells

The stems and committed cells of normal tissues:

When there is chronic inflammation, it is a true sign of precancer, indicating that there is hepatocellular cancer which is infected by hepatitis B or C viruses. This possibly results in a high probability of liver cancer in human (4); besides, Helicobacter pylori infection augments the growth and spread of human stomach cancer (5). The stem cell which is the first structure of most organs can very easily be reproduced infinitively and can have several separate differentiations (6-8). The number of stem cells is very low, although it never comes to an end; they are usually placed in suitable places to protect against external influences (9). A committed precursor or a cell amplifier which makes proliferation in a tissue is the next step in the differentiation of stem cells. The characteristics of this partition include continuous replication, the ability to differentiate to a limited extent and a partial reproducibility. At this stage, the stem cells are exposed to such regulatory factors as hormone and growth factors which control their multiplication of the tumor cells

1.1.2 Genetic predisposition or genetic precancer

It is highly probable that because of one or several mutations, a tumor is a genetically transformed clone of cells with fixed pathological features. This is supported by hemoblastoses or hereditary forms of cancer. It is most obvious that hereditary cancer is shown by the malignant tumor of the ocular retina or retinoblastoma (10) which is the result of a recessive mutation hereditarily derived from the parents. With only one copy of Rb, the normal cellular phenotype can be preserved; nevertheless, with an irregular mutation in the ocular retina cells which inactivates the other Rb allele and results in visual aspects of retinoblastoma in early childhood, it becomes clear that the common characteristic of almost all cases of hereditary retinoblastoma is posing damage to both eyes. (11). Therefore, the preliminary mutation proves that the precancer which can be identified in crowded groups of cells carry other mutations.

There is a very common case for the genetic changes and it is that of breast cancer (12). This cancer is partially induced by such genes as BRCA 1 and 2. Here the mutation frequency is associated with the occurrence frequency of this cancer. It has not yet been proven how BRCA1 and 2 have a relation with the appearance of mammary gland cancer; nevertheless, it is believed that genetic factors may have a role in the emergence of this tumor in some populations.

1.1.3 The role of inflammation in development of precancer

There has been some investigation into the role of inflammation in tumor growth so far (2); however, there seems to be a grave problem, The first one is that inflammation includes large groups of cells, whereas tumors are monoclonal.; second, tumorigenesis is underlain by pathology of individual cell mutations which are not linked with inflammation. There are, however, three connecting paths which should be taken into consideration between malignant growth and inflammation. Firstly, the stem cells are stimulated to multiply, flowing which the tumor stroma is produced. In this way, sufficient extracellular matrix is created to invase and metastasize (13); secondly, the formation of microcirculation, or angiogenesis, is required for the respiration and nutrition of the tumor and the deletion of the products of its essential activity. Finally, cytokines, or tumor growth factors generation is needed for tumor growth (14-18).

1.2 Cell cycle

All living organisms on the earth ranging from unicellular to multicellular mammals have been exposed to repeated cell growth and division for over three billion years. According to the German pathologist Rudolf Virchow's cell doctrine in 1858, a cell arises from a previous cell exactly like the animals and plants which originate from previous animals and plants. Cells are derived from the cells which already existed and it is by division that more cells can be produced.

The cell cycle is a process in which there is cell growth, division of cells into identical daughter cells and DNA duplication. In the classification of the cell cycle, there are two special parts, namely interphase and mitosis (Figure 1). The length of the cell cycles variably depends on the organisms and the cell types. It is estimated that cell division occurs over a period of twenty four hours (19).

Figure 1: Phases of the eukaryotic cell cycle.

There are four phases for cell cycles which are as follows:

G1 phase

The first phase is called gap phase 1 which is usually the longest and variable in length. This phase begins when mitosis and cytokinesis are completed; it continues up to the beginning of S-phase. In the first phase, the cell multiplies its DNA or leaves the cycle in order to go into an inactive state (i.e. G0- phase)

S phase

The replication of chromosomes is limited to S-phase (DNA synthesis phase) of interphase; this usually takes six hours. This phase is, in fact, the main initiation of the synthesis of DNA and it happens several hours after the cell started DNA synthesis. In S-phase, each chromosome multiplies, making a pair of sister chromatids and also centrioles.

G2 phase

The final and usually the shortest phase in interphase is called G2-phase in which the cell experiences fast growth so that it can be prepared for mitosis. This phase continues until chromosome replication and the DNA synthesis during the S-phase is successfully completed. The duration of this step is often four to five hours. It is difficult to distinguish the multiplied chromosomes one by one because they are in the form of chromatin fibers which are loosely packed. In this phase, the cell is prepared for mitosis (M-phase) which is initiated by prophase.

At the end of G2-phase, a control checkpoint (G2 ) can make it clear which cells can continue to go into M-phase for division and which cells with damaged DNA will be prevented from going into mitosis. This checkpoint can also help the cells sustain genomic stability and prevents the damaged cells from spreading; this phase is very important in learning about the molecular causes of cancer.

M phase

The M-phase includes the overlapping of mitosis and cytokinesis processes. Mitosis has five levels, including prophase, prometaphase, metaphase, anaphase, and telophase. The start of cytokinesis is during the anaphase and its termination is at the completion of mitosis. When cytokinesis finishes, the parent cell has (20, 21). Its two G1 phase progeny and it is time for the cell to repeat the cycle.

2. Breast Anatomy and Physiology

The nipples are the milk ducts which open and the areola is the dark circular area around the nipples. These two are the Montgomery's tubercles consisting of the opening of sebaceous and sweat glands (i.e. Montgomery gland) which secrete lubricating substance for the nipples. The following picture shows a profile view of the anatomical structure of breast:

Profile view of the anatomical structure of breast

The place of the lactiferous sinuses is under the areola of the breast from which milk secretes through the nipple or may enter the Montgomery's tubercles.

The gland cells around the central duct produce milk. The gland cells which are surrounded by myoepithelial cells are contracted and as a result of which milk is injected into the milk duct and then transferred into the lactiferous ducts and finally into the lactiferous sinuses.

A cross-section view of the alveolus

When an infant breastfeeds, he pulls the nipple and the areola into his mouth and this causes the nipple to stretch two times more than its normal length. About 0.03 seconds after the maximum elongation of the nipple, milk is ejected.. (22).

Ramsay said that when the infant's gum squeezes the areola, it causes oxytocin to be released and then there will be an increase in the diameter of the milk ducts and so there is a faster movement for the milk fat globules toward the nipple. After suckling begins, milk ejection happens on a average of 50 seconds. The number of milk ejection that a woman has during breastfeeding varies from 1 (26%) to 2.9 (74%). Generally speaking, the mean number of milk ejection is 2.5 ejection /breastfeed (23).

3. Breast Histopathology

4. Breast Cancer Epidemiology

Breast cancer has a wide distribution among different groups. The percentage of breast cancer is higher and the Caucasian race is more exposed to breast cancer in comparison to other ethnic groups in the United States. In this part the incidence of breast cancer will be described in terms of age, ethnicity and geographical variation.

4.1 Incidence by Age

The risk model is set according to the population averages. Irrespective of degree of risk (high or low) breast cancer risk for women relies upon such factors as family history, age menstruation, genetics and other unknown factors. Although breast cancer is less common at the young age of thirties, younger women are more vulnerable to suffer from breast cancer than older women

4.2 Incidence by Ethnicity

All women are exposed to develop breast cancer and as they get older, the chance of developing breast cancer gets higher. About 77% of breast cancer can be found in women over 50. Based on the findings of the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute, White, Hawaiian, and African-American women are at the highest (four-fold) risk of invasive breast cancer among the minority groups in the U.S. whereas Vietnamese, American Indian and Korean women have the lowest risk of being exposed to breast cancer in the U.S.

It was proved African-American women at the age groups of 30-54 and 55-69 had the highest level of death rates due to breast cancer. Following them were the Hawaiian and white non-Hispanic women. It was also revealed that white women of 70 had higher death rates than African-American women.

4.3 Incidence by Geographical Variation

The incidence of breast cancer is different for different countries which may be originated in the type of dietary habits, number of pregnancies and also cultural differences.

The vastness of geography can prove which cancer is not an intrinsic result of life, but it is a consequence of environmental factors, life style and heredity. In 1981 Doll and Peto showed the percentage of people who suffered different types of cancer in countries with the highest and lowest levels of risk. Based on their findings, Doll and Peto expressed that around 75 to 80 percent of cancer cases which occurred in the U.S. in 1970 could potentially be avoided. Their analysis did not indicate the reason why a special cancer is prevailing or not in a certain region; it examined all the data available from analytic studies, clinical medicine and basic research to illustrate the number of cancer related to nutrition, alcohol consumption, smoking and other factors.

The studies conducted on the migrants who moved from higher to lower risk areas and vice versa proved that many types of cancers can actually be avoided. The fluctuation in the incidence of some types of cancer is much more evident immediately after migration than in other occasions. For instance, it was observed that the low occurrence of colon cancer which is common in Japan sharply went up in the Japanese who moved to Hawaii or other continental United States. Nevertheless, it can be seen that the next generation of the Japanese who migrated to the U.S had a higher rate of breast cancer in comparison to their predecessors. Finally, the occurrence of breast cancer among Japanese American women is about equal to or exceeds the rate among the white women of the United States (24, 25).

5. Breast Cancer Etiology

All over the world, breast cancer is the most common type among women and in 2002, it was responsible for 203,500 and 39,600 for new cancer diagnoses and deaths. Since 1997 there has be a sharp fall in mortality from breast cancer and it can probably be attributed to the therapy with tamoxifen and possibly other kinds of chemotherapy (27). Based on the evidence, the hormonal etiology of breast cancer is in alignment with the idea that the main stimulant for the spread of breast cell is estrogen (28, 29) and progesterone accounts for the growing rate of cancer spread. (30).

5.1 Reproductive Factors

There are some prevailing factors which account for the breast cancer; they include early age at menarche, late age at menopause, late age at first full-term pregnancy and finally weight. The age curve for the cancer incidence of breast cancer demonstrates the significance of ovulation in the determination of risk (31).

Nowadays one of the risk factors for breast cancer which has been agreed on is early age at menarche (29). As a rule, the risk of breast cancer declines by 20% for each year when menarche starts with delay. According to one study, young women who have early menarche (at 12 or earlier) and their regular cycles are rapidly established have about fourfold increase of risk for breast cancer compared to the women whose menarche is delayed (age 13 or later) and have long duration of irregular cycles. (32).

Some recent studies have revealed that personal lifestyles such as leisure time, exercise and activity can significantly affect breast cancer risk both in young women (<40 years of age) (33) and older, postmenopausal women (55 to64 years of age) (34).Although many researchers have shown that there is an association between a decrease in breast cancer risk and an increase in the mount of exercise, some have not come to this conclusion. (35).

5.2 Weight

There is a relation between weight and the risk of breast cancer. This factor highly relies on age. For each extra 10 kilograms of bodyweight in postmenopausal, there is on average 80% increase in their risk of breast cancer (36) and that is because of higher circulation of estrogen due to the transformation of the adrenal androgen androstenedione to estrogen by the aromatase enzyme which is present in body fat. On the contrary, this relation can hardly be established in premenopausal women (37).

5.3 Age at First Birth

When the first birth is at an early age of 20, the woman's risk of breast cancer decreases by 50% but full-term pregnancies at later ages add a benefit to protection. (38). The women who have their first full-term pregnancy very late are actually more exposed to breast cancer than nulliparous women. Many studies on epidemiology have confirmed the effect of a late full-term pregnancy.

Based on the above-mentioned findings, it seems that a first pregnancy can lead to two opposite effects on the risk of breast cancer; one of them is a short-term increase of risk and the other is a long-term decrease in risk (38).

The amount of estradiol in the first trimester quickly goes up in comparison to the subsequent pregnancies (39). As a result, the effect of estrogen exposure to the breast in early pregnancy involves a higher risk. However, this negative impact of early pregnancy on breast cancer risk can be suppressed by two useful hormonal consequences of completing the pregnancy. Based on previous studies, in comparison to nulliparous women, the levels of prolactin ( a polypeptide hormone) in parous women is noticeably lower (40, 41). Prolactin is responsible for the regulation and enhancement of the estrogen impacts on the breast tissue. Furthermore, it has been proved that parous women have lower levels of bioavailable estradiol than nulliparous women (42). From a molecular point of view, the hormonal alterations in pregnancy may induce unreturnable differentiation and apoptosis in some cells which have already collected one or more related somatic mutations essential for the development of breast cancer.

5.4 Exogenous Hormones

It is believed that hormone oral contraceptives and replacement therapy are exogenous rather than endogenous hormonal exposures which women can experience; hence, they are considered as possible factors for breast cancer risk.

5.4.1 Oral Contraceptives

Many articles have focused on the application of OC in breast cancer risk. There have been 54 studies done on more than 150,000 women who used combination oral contraceptives (COCs) which had an estrogen and progestin in combination in a single pill. The result of the analyses proved that there was a risk of cancer among them (43). Accordingly, there is a moderate increase in the risk of breast cancer among the current and recent users of COC with relative risk (RR) = 1.24) and (RR = 1.16), respectively.

5.4.2 Hormone Replacement Therapy

There is a direct relation between the increase of breast cancer risk and hormone replacement therapy. Another analysis on 51 studies which covered 160,000 women revealed that as the duration of use increases, the risk of breast cancer rises (44). The women who used HRT during the five years of diagnosis had a higher rate of risk by 2.3% per year while those who had stopped using HRT five years or sooner regardless of the duration of use had a minor and non-significant increase in breast cancer risk. Most of the data in this analysis is about estrogen-only replacement therapy (ERT). Combined hormone replacement therapy (CHRT) which has progestin with estrogen on a continuous basis during a monthly cycle has gained rapid popularity in the past two decades; however, the risk of breast cancer with CHRT is higher than that in ERT (45). There has been a large-scale research on the relationship of CHRT and breast cancer reported by Ross and his colleagues, who found out that the degree of risk for each five years of use was about four times higher for CHRT than for ERT users (46).

5.5 Genetic Determinants

It has been proved that there is a relation between family history of breast cancer and a high degree of risk being exposed to this disease. This is especially applicable when a woman had a bilateral disease or was affected at an early age. It was observed that there is an increase by two or three times and nine times in the first-degree relatives of women with breast cancer and first-degree relatives of premenopausal women with bilateral breast cancer respectively.

During the last eight years two genes, namely BRCA 1 and BRCA 2 have been examined in families with high risks (47, 48). The first estimation of penetrance of these genes were reported to be high, but when more selected families were examined, the risk of breast cancer in women with a BRCA gene mutation seemed to rank less than 50 percent (49, 50). Another study was on more than two hundred different mutations in the BRCA genes and two common mutations of BRCA1 were identified in the subjects of Ashkenazi Jewish descent: 185delAG (0.9%) and 5382insC (0.13%) (51), but in BRCA2, the 6174delT mutation was (1.5%) of the Ashkenazi Jewish population, and the 999del5 mutation was (0.6%) of the entire Icelandic population (52).

Extra high-familial-risk genes such as germ line p53 mutations (LiFraumeni syndrome), which are quite scarce, suggest that there is another possible mechanism for this syndrome to be genetically susceptible to breast cancer, which is a common issue (53).

Some common allelic variations in estrogen metabolism genes like cYP17,CYP19 and HSD17B1 are being examined to detect their role in breast cancer risk (54). It has been reported that common sequence variants in CYP17 and HSD17B1 augment the risk of advanced breast cancer (55). These variants in other candidate genes have not been multiplied because they needed large sample sizes and it was necessary to have a more thorough understanding of the underlying haplotype structure of these genes in the human population. The additionally significant sources of such candidate genes include genes encoding protein in intracellular pathways, genes in the growth factor pathway, DNA repair and steroid receptor transactivation (55).

6. Breast Cancer Histopathology

Although breast cancer can start in any part of a breast tissue, the majority of breast cancers begin in ducts, a small percentage starts in the lobules and even a very few number of them can initiate in other tissues of the breast. Based on different features of a particular disease, there are different types of breast cancer. One type is named according to the origin where it started to appear such as invasive ductal carcinoma. Another type is named with respect to its shape under microscope as in tubular carcinoma, which looks like tube-shaped cells. Every one of these types has its own distinctive prognosis and symptoms.


Invasive (or infiltrating) ductal carcinoma:

This is the most prevailing kind of cancer (about 70% ) and initiates in the duct and then enters the duct's wall finally penetrates the fatty tissue of the breast. Thus, it has the capability of spreading (metastasize) throughout the body via lymphatic system and bloodstream.

Invasive (or infiltrating) lobular carcinoma:

The second very common tumor type which accounts for 10% of all breast cancer starts in the terminal ducts of the glands which produce milk

Medullary carcinoma:

There is another type of cancer with 3%-6% occurrence. It happens for the women who are genetically predisposed to breast cancer. According to different studies, about 13% -19% of all medullary carcinomas can be found in the women with a BRCA1 mutation. The defining line between the cancer tissue and the normal tissues is almost well set. All in all, the patients with medullary carcinoma are better diagnosed than the patients with other types such as ductal or lobular carcinoma.

Paget's disease:

In this type of breast cancer in which the nipple and areola are at the 3% risk in comparison to all other breast cancers, it is often related to abnormal redness and scaling of the skin of the nipple and areola and in this case, women suffer burning or itching. Paget's disease may not change its situation or can be related to invasive cancer.

Inflammatory breast cancer:

This kind of cancer is named so due to the initial symptoms which are warmth, redness and swelling of the skin of the breast without a distinct lump for only about 1% compared to other types of breast cancers. The emergence of infection or inflammation results from the cancer cells which block the path of lymph vessels or channels in the skin over the breast.

Invasive ductal carcinoma.

Another type which is very common among older women with 3% of breast cancers is mucinous or colloid carcinoma. Papillary carcinoma and tubular carcinoma each indicates around 1% of breast cancer prognosis. The degree of diagnosis for mucinous and tubular carcinomas is well ahead of the more common type of invasive ductal or lobular breast cancer. Adenocystic and carcinosarcoma breast cancer is responsible for 0.4 and 0.1 percent of all cases breast cancer (57-61).

6.1 Staging and Grading Breast Cancer

This stage displays the condition of disease or biologic potentiality for a patient's tumor. Staging follows these three aims: (a)therapeutic way which is appropriate for the patients, (b)the prediction of the status of the disease and (c) comparison of the results gained from different sources through different means. (62).

The classification which is a world standard and approved by the American Joint Commission on Cancer Staging is the TNM classification of the International Union Against Cancer (UICC)

Knowing the stage and grade of tumors will help doctors precisely decide what treatment is suitable for the patients. There are three levels for the stage of breast cancer, namely tumor, lymph nodes and Metastasis. These factors are called TNM classification.

The TNM relies upon the clinical features of tumor (T), the place of lymph nodes (N) and metastases (M). The qualification of a tumor is established by its size, so a T1 is a tumor less than 2 cm, a T2 is 2 to 5 cm, and a T3 is more than 5 cm. Similarly, N0 indicates negative, normal and regional lymph nodes. Generally, lower numbers show that cancer is less serious (63).

6.1.1 Stages of Breast Cancer

By determining the TNM classification, researchers can put cancer in four major stages which are used by American Joint committee on Cancer staging system.

Stage 1 of breast cancer or early breast cancer shows that tumor size is less than 2 cm m across. It does not spread to the other parts of the body nor does it go to the lymph nodes.

Stage 2 indicates that the tumor size is between 2-5 cm; it can affect the lymph nodes but does not spread to the other parts of the body. If tumor size is 2 cm, it is often called early breast cancer, but if the tumor size is closer to 5 cm, it is usually considered as locally advanced breast cancer.

Stage 3 of breast cancer means the tumor size is bigger than 5 cm across; it also implies that the lymph nodes are affected by cancer in the armpit but it has not spread to the other parts of the body. This step is also known as the locally advanced breast cancer.

Stage 4 of breast cancer refers to the fact that tumor has varying sizes and can affect the lymph nodes and spread to other parts of the body. This stage is known as metastatic or secondary breast cancer (64).

6.1.2 Grading of Cancer

The grade represents the idea concerning the degree of resemblance of the cancer cells to the normal breast cells under microscope. Researchers usually designate certain grades to cancer such as low grade, high grade or a number between one and four. Low grade cancer cells look like normal cells and high grade cancer cells differ from the normal breast cells. (65).

There is another system of classification known as the Columbia Clinical Classification (CCC) which has been defined by Haagensen. Despite the fact that this system is much easier than the TNM, it is less precise as a classification system; stage A shows a tumor is confined to the breast; stage B includes tumors and axillary lymph node enlargement; stage C shows the presence of dangerous diagnostic signs in breast; and stage D represents metastatic disease. Although both classifications basically explain the same procedure according to the related stage, the results of UICC-TNM system can be universally interpreted (66).

6.2 Breast cancer recurrence

Recurrence accounts for the returns of breast cancer after the initial treatment. There are three kinds of recurrent breast cancer:

6.2.1 Local recurrence:

The first type happens when cancer cells emerge at the original tumor site because the initial treatment fails to work. The remained parts of the breast skin and fat after mastectomy make local recurrence possible, although it is unusual. The women who are treated with breast conserving therapy and radiation face a higher risk of recurrence of this cancer in comparison to others. The treatment of the local recurrence of breast cancer relies upon the first therapy in the first prognosis. If breast conserving surgery is initially performed, the recurrent breast cancer will usually be treated with mastectomy.

6.2.2 Regional recurrence

The second type is the regional recurrence of breast cancer which occurs in the chest muscles, in the internal mammary lymph nodes under the breast bone and between the ribs, in the nodes above the collarbone and in the nodes surrounding the neck. The last two places account for more critical cancers. Generally, the regional recurrence is quite common and its percentage of occurrence is 2%-5% in comparison to all breast cancer cases. However, its treatment is complex and involves surgery for the removal of the cancerous node, radiotherapy, and chemotherapy chemotherapy and adjuvant endocrine therapy considering the type of the previous treatment already applied.

6.2.3 Distant recurrence

The most critical type of recurrence with lower survival is known as metastasis. In this type, the cancer leaves the breast tissue and usually goes to the axillary lymph nodes. In 65 -75 percent of breast cancer, it spreads from the lymph nodes to the bone. It hardly ever happens that the breast cancer metastasises to other parts such as the lungs, brain, liver or other organs. Since the cancer is not limited to one fixed area, surgery can hardly be a solution to metastatic breast cancer. However the kind of treatment which is employed includes chemotherapy, radiation therapy or endocrine therapy. (67-72).

It sometimes happens that a new cancer occurs some years after the initial tumor in a different area of the breast, but with different pathology and not related to the first type of cancer. This is, however, considered as a new type of cancer and is not regarded as a recurrence. (67).

Of course, five years after surgery and without additional therapy, 60% of the women survive the breast cancer (72). If no further treatment is applied, it is most probable that breast cancer will recur during the first two years. (73).

The findings of 55 clinical experiments on breast cancer recurrence which involved 37,000 patients confirmed the clustering of recurrence risk some years after the first prognosis of early breast cancer for the patients who had not received adjuvant endocrine therapy. During the first 10 years after diagnosis, the death rate related to the incidence of recurrence and breast cancer went up. The patients who did not receive adjuvant hormonal therapy were reported to have recurrence rate of nearly 50% and 32.4% in node-positive patients and node-negative patients in the 10 years after diagnosis, respectively (74).

Unlike other types of different cancers, it is believed that breast cancer cannot be cured if recurs during the first five years????????????. Although it may recur after ten or twenty years following the first diagnosis, the risk of recurrence decrease over time (73).