Almost all breast cancer patients receive radiotherapy after undergoing breast conservation or mastectomy. The benefits of radiotherapy on long term survival may not essentially be free of complications. Patients may have a wide range of normal tissue reactions, and tissue toxicity may be in the form of asymptomatic changes in tissue structure and function. (Bentzen et al, 2003). The outcome of radiotherapy can be separated into early/acute or late, depending on whether they arise within or after 90 days after radiation treatment (van der Kogel 1993). Radiotherapy significantly reduces the risk of local recurrence; however it does cause an increase in cardiac disease, which is similar to that observed in the older patients. Many of the techniques used engross unavoidable irradiation of the heart resulting in a 27% increase in mortality from heart disease and potentially attenuating the beneficial effect of radiotherapy on overall survival (Clarke et al, 2005).
In this study an attempt will be made:
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To predict late normal-tissue reactions by genotyping single nucleotide polymorphisms (SNPs) in the ATM gene of 642 breast cancer patients, post radiotherapy. This may help determine if it is possible to predict which individuals are predisposed to radiotherapy complications.
Look for evidence of cardiac damage in six breast cancer patients having radiotherapy, at baseline and six weeks post completion of radiotherapy. This will be measured in vivo with cardiac MRI and correlated with surrogate in vitro markers of biological ageing.
To determine whether radiation induced long-term changes in miRNA profiles can provide a mechanistic link between radiotherapy, biological ageing and an increased risk of heart disease.
1.1 Breast Cancer
Cancer is a disease which involves abnormal changes in the genome. This abnormality takes place due to mutations which generate oncogenes with dominant gain of function and tumour suppressor genes with recessive loss of function. Weinberg.R et.al (2007). Cancers arises from accumulation of genetic mutations in genes that are involved in DNA repair and genes that control cell growth, this leads to cells growing and dividing in an uncontrollable manner to form a tumour. Generally, these genetic changes develop during an individual's lifetime and are found in certain cells. These changes are known as somatic mutations and they transpire in oncogenes and tumour suppressor genes which have been reported to be a risk for the development of breast cancer. Chanock S.et.al (2007). In less frequent circumstances, gene mutations which have been inherited from a parent enhances the risk of developing cancer. Those who have these inherited genetic changes, additional somatic mutations in other genes is required to occur for cancer to develop. Hereditary cancers are related to inherited gene mutations. Hereditary breast cancers have a tendency to occur earlier in life than non-inherited cases and are more likely to involve both breasts.
BRCA1 and BRCA2 are important genes implicated in hereditary breast cancer. Those who have inherited certain mutations in these genes have a high risk of developing breast and ovarian cancer. Inherited changes in other genes, including CDH1, PTEN, STK11, and TP53, have been found to increase the risk of developing breast cancer. Inherited variants of the ATM, BARD1, BRIP1, CHEK2, NBN, PALB2, RAD50, and RAD51 genes, are also associated with breast cancer risk.
Figure 1: A diagram to show the genes associated with the development of breast cancer when mutated.
Table 1: A table to show the genes associated with breast cancer when mutated
Breast cancer is the most common form of cancer in women, affecting one in nine women. Each year almost 44,000 women develop this disease in the UK, and more than 12,000 die from it. Evans D. et.al (2007). As a woman ages, the likelihood of developing breast cancer increases. This may be a result of genetic damage that transpires during the course of life. Ronckers C et.al. (2005). The causes of breast cancer are not known; yet epidemiological research into the aetiology of breast cancer has largely focused on adult life risk factors. Many studies have shown that women with risk factors are more likely than others to acquire breast cancer. Risk factors include; age, gender and family history of breast cancer.
Radiotherapy is commonly applied during cancer treatment and involves cancer cells being destroyed by beams of high-energy X-rays. These beams damage the DNA of tumour cells and therefore sustain their potential in reproducing, and this accomplishes the key aim of radiotherapy, which is to damage all cancer cells completely. The radiotherapy dose is important and therefore is adequately measured so damage to surrounding tissues is kept to minimum. The key advantage for radiotherapy is that it is a focused treatment for malignancies and the treatment x-rays are aimed directly at the tumour site with fewer side effects on the rest of the body. It also avoids prolong general anaesthesia and there is a lower risk of systemic complications and lastly it increases the ability to preserve normal anatomic structures. The types of radiation treatment include; external beam radiotherapy, interstitial brachytherapy and unsealed radionuclide therapy.
Radiotherapy and DNA damage
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Radiation has the ability to interact with different molecules and damage them. As the human body stores large volumes of water, there is a greater possibility of radiation interacting with water and producing free radicals (hydronium (H.) and hydroyxls (.OH) ).Free radicals can produce compounds, which cause chemical toxicity. These compounds are hydrogen peroxide (H2O2). DNA repair methods aim to defend the
body against these toxins, although a small part of the damage is not repaired or is repaired improperly. In both circumstances, the damaged DNA can be repaired by several mechanisms, these include; direct chemical reversal of the damage, base excision repair, nucleotide excision repair and mismatch repair. If the damage is not repaired, the cells will undergo apoptosis. There are cases where the cell survives and functions abnormally due to the damaged DNA. The different types of DNA damage are damaged bases and single or double strand breaks.
Figure 2: To show the effect of radiotherapy on DNA Mohammad A.et.al (2007)
1.2.1 Radiotherapy complications
Radiotherapy has improved breast cancer survival but some have known to develop complications, which are either local (skin and subcutaneous) or involving deeper organs (lung and cardiac). There are many complications associated with breast cancer radiotherapy and these are outlined in figure 3.
Figure 3: A diagram to demonstrate the complications that are gained after receiving radiotherapy for breast cancer. Jassem J.et.al (2006)
1.2.2 Can long term radiotherapy damage be predicted?
Adverse reactions to radiotherapy is a major clinical problem and difficult to predict. Cancer patients react differently with respect to their normal tissue response to radiotherapy. If this variability could be taken into account during treatment planning, the therapeutic strategy could be individualised. Therefore being able to predict a patients reaction to radiotherapy is currently a sought after goal. Previously, studies have focused on designing assays regarding cellular radiosensitivity or sub-cellular damage end-points. However, recent studies are focusing on the concept that normal tissue radiosensitvity could be predicted from individual genetic profiles.
Recent technological advances are being used to uncover the molecular basis of normal tissue complications, and provide a list of radiosensitivity candidate genes. Micro-array based gene expression profiling can determine expression patterns before and after irradiation of cells or tissues and new radiation induced genes could be identified. Andreassen C (2005).
Skin and Cardiac damage after Breast Cancer radiotherapy
Radiotherapy reduces the risk of local recurrence after surgery, with a reduction in cancer mortality offset by an increase in contralateral breast cancer and cardiac disease, which is observed in older patients. Most of the techniques that have been used, incur irradiation to the heart, these results in increase in mortality from heart disease, and reduces the benefits of radiotherapy on survival rate Tanteles G.et.al (2009). The cardiovascular mortality risk in patients who receive radiotherapy for left-sided breast cancer, is observed after 10 years. This source of risk may be due to the side and technique of irradiation of the left anterior descending artery. Studies have also been carried out to show that women who have received adjuvant radiotherapy for left-sided lesions have an increase risk of myocardial infarction. Tanteles G.et.al (2009). Many of the cardiac structures including myocardium, valves and coronary arteries are sensitive to radiation and prone to radiation damage. Stewart et.al. (1995).
There are additional risk factors associated with an increased risk for cardiovascular morbidity and mortality after breast cancer radiotherapy. These include irradiated heart volume, total radiation dose and fractionation. Tanteles G.et.al (2009).
Figure 4: Cardiac damage after radiotherapy. Tanteles G.et.al (2009).
Radiation induced heart disease, results from microvascular and macrovascular damage. The damage to the microvascular component begins by endothelial cell damage within cardiac structures. This leads to ischaemia, progressive obstruction of the vessel lumen. The damage area is then replaced by fibrous tissue. Radiotherapy for both left and right sided breast lesions, causes the heart to receive over 1 Gy dose from scattered irradiation Li et.al.(2000). This exposure to the heart, leads to cardiac damage, resulting in cardiac mortility. An example includes those involved in the atomic bombings in Hiroshima and Nagasaki, the survivors showed evidence of this. Tanteles G.et.al (2009).
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Tanteles G.et.al (2009) examined the relationship between the late normal-tissue radiation injury phenotypes in 149 irradiated breast cancer patients and the development of post-radiotherapy cardiovascular disease. The association they concluded from the results show that telangiectasiae could be used as a marker of future radiation-induced cardiac complications.
Figure 5: Patient cohort to show the relationship between late normal-tissue injury phenotypes in 149 irradiated breast cancer patients and the development of post-radiotherapy cardiovascular disease. Tanteles G.et.al (2009)
Telangiectasia is small dilated blood vessels commonly observed on the skin and mucous membranes. Common causes of telangiectasia include alcohol use, ageing and sun exposure. One of the key diseases associated with telangiectasia is ataxia- telangiectasia. Telangiectasia can develop at six months to many years after the completion of radiotherapy, they occur most commonly in the boost area. The initial effect of radiotherapy is DNA damage and the most vulnerable cells are endothelial cells. Telangiectasiae is formed by impaired tissue microcirculation drainage leading to stasis in the collecting venules, resulting in capillaries and venules expansion Tanteles G.et.al (2009).
Figure 6: A clinical view of dilated blood vessels typical of telangiectasia.
ATM (ataxia telangiectasia mutated) gene
Recent studies have shown patients with ataxia telangiectasia have extreme levels of sensitivity to radiotherapy. In the 1970s it was established that this sensitivity may be linked to mutation in various genes, such as the ATM gene. Many of these studies have investigated potential genetic markers for predicting normal tissue toxicity after radiotherapy.
ATM (ataxia telangeictasia mutated) protein plays a crucial role in the detection of DNA damage and activation of pathways that result in cell cycle arrest, DNA repair or apoptosis Kastan MB.et.al (2000). Mutations in the ATM gene cause ataxia telangiectasia. The mutated ATM gene therefore produces proteins that do not function properly. This results in the cells being extremely sensitive to radiation and do not respond correctly to DNA damage. Those who have mutations in one copy of the ATM gene, particularly those who have at least one family member with ataxia-telangiectasia, may be at an increased risk of developing breast cancer.
Table 2: List of studies associating the ATM gene with radiation-induced morbidity. Studies are listed with tumour type, number of patients included, the endpoint studies, the mechanism(s) by which the candidate gene is involved in the pathogenesis of normal tissue toxicity.
Biological ageing and radiotherapy
There has been improved breast cancer survival following radiotherapy, however there has been an increase in cardiovascular deaths following radiotherapy. This is due to radiation damaging heart tissues.
Biological ageing is the progressive decline in physiological ability to meet demands that occur over time. It is due to the accumulation of damage at the cellular level and the rate of biological ageing is determined by both environmental and genetic factors Adams.J.et.al (2004).
Figure 7: A figure to show the five theories associated with ageing.
1.4.1. In vitro assays and biological aging
Telomeres are sequences of non-coding DNA situated at the ends of the chromosomes and have a great impact in cellular ageing and replication. When DNA damage occurs, DNA repair mechanisms do not act on telomeres and therefore the damage remains unrepaired. As damage builds up and strand breaks occur, the length of the telomere begins to shorten. Telomere length has been associated with morbidity and mortality and is considered to be a marker of the cumulative damage that a cell has been exposed to and has been thought to be a biomarker of biological ageing. Adams.J.et.al (2004).
It has been obsereved that DNA damage, response proteins are involved in telomere maintenance, which suggests that telomere shortening may be related to breast cancer susceptibility. Short telomeres may be able to show an increase in cell turnover or loss of important genes in the telomeric region, which could lead to a failure of apoptosis. Kim.et.al (2002).
The cellular approach involved with damaged DNA entails injured DNA recognition, a period of damage assessment which is imposed by checkpoints, mechanism by which the cell actively halts progression through the cell cycle until it can ensure that an earlier process, such as DNA replication or mitosis, is complete. G1 and G2 are the two cell cycle checkpoints. Hartwell and Kastan (1994).
Apoptosis is an important mechanism for eliminating cells with DNA damage. Defects in apoptotic pathways have been known to contribute to a number of human diseases, ranging from neurodegenerative disorders to malignancy. Lowe.S.et.al (2000). During apoptosis, intracellular material is degraded and the cell breaks into fragments surrounded by membrane. The assay for quantifying apoptotic response in peripheral blood lymphocytes is based on irradiating lymphocytes in short term culture and assessing DNA content by flow cytometry.
Studies have shown that apoptotic response to DNA damage reduces with ageing. In a study conducted by Polyak et.al (1997), there was evidence of reduced apoptotic response of lymphocytes to a 5Gy radiation exposure in mice with increasing age. The ability of peripheral blood lymphocytes to undergo radiation induced apoptosis in vitro falls by 0.5% per year. In a study conducted by Docherty et al (2007) it was established that in vitro apoptotic response to ionising radiation in peripheral blood lymphocytes fell by 15% in 12 breast cancer patients, when repeated one year post radiotherapy. This is 30 years of biological ageing over a one year period post irradiation.
Figure 8: Diagram illustrating an individuals PBL decreased capability to undergo apoptosis.This represents a biological ageing effect of 30 years in a one year period. (Docherty et al (2007)
The alkaline single-cell microgel electrophoresis (Comet) assay is a technique used to detect DNA damage and repair. It involves the encapsulation of cells in agarose suspension, lysis of cells in neutral or alkaline conditions, and electrophoresis of the suspended lysed cells. This is followed by visual analysis with staining of DNA and calculating fluorescence to detect DNA damage which includes single strand breaks, double strand breaks and alkali-labile sites.
In a study conducted by Vrhovac.V.et.al (2003), alkaline comet assay was used as a biomarker of exposure to observe the ongoing exposure to ionising radiation of 50 medical workers exposed to ionising radiation and 50 unexposed individuals were used as a control. The primary DNA damage was observed by measuring the extent of DNA migration in peripheral blood leukocytes. The results showed that those who were exposed to radiation for different periods of time, demonstrated increase levels of DNA damage in comparison to the controls. The results confirm that using comet assay can be an additional complement to standard observations of DNA damage.
Figure 9: Atrial cardiomyocyte nuclei after comet assay. (A) Nucleus with undamaged DNA. (B) Nucleus with approximately 50% of DNA in the tail. Lenzi.P.et.al (2003)
1.4.2 Cardiac function by MRI
Magnetic resonance imaging (MRI) is a noninvasive test that creates detailed images of organs and tissues using radiowaves and magnets. Cardiac MRI uses a computer to create images of the heart as its beating, producing both still and moving pictures of the heart and major blood vessels. It is used to look at the structure and function of the heart, so the ideal treatment can be given to those with heart problems. Cardiac MRI is a common test for diagnosing and evaluating a number of diseases and conditions, such as coronary artery disease and congenital heart defects. Cardiac volume in the radiation field can be measured using CT scanning and subclinical cardiotoxicity can be determined by MRI.
Figure 10: Figure A shows the heart's position in the body and the location and angle of the MRI images shown in figure C. Figure B is a MRI angiogram. Figure C shows MRI pictures of a normal left ventricle (left image), a left ventricle damaged from a heart attack (middle image), and a left ventricle that isn't getting enough blood from the coronary arteries (right image). www.heart.org.in/diseases/cardiac-mri.htm
1.5 miRNA and cardiac damage
miRNAs are short, non coding RNA molecules, of approximately 21 base pairs. They are negative regulators of gene expression and inhibit mRNA translation, or promote mRNA degradation. It is this regulation and deregulation that has been shown to influence the development of normal cells into diseased cells. Being able to understand how miRNA expression influences the development will lead to improved diagnosis and treatment for heart disease.
The possibility that microRNAs might be involved in heart disease was first suggested by the discovery of distinctive patterns on microRNA expression in the hearts of normal mice and mice that suffered from heart disease. Rooij.E et.al (2006). Certain microRNAs were upregulated and others were downregulated in mice subjected to thoracic aortic banding. Also, many of these microRNAs were dysreguated in failing human hearts, suggesting they established a diagnostic molecular signature for cardiac pathogenesis. Rooij.E et.al (2006). This shows that microRNAs are in fact involved in cardiac function. Rooij.E.et.al (2008) showed that miR-1, miR-229, miR-133, and miR-150 were found to be downregulated during pathological cardiac remodelling, whereas miR-21, miR-23a, miR-125, and miR-195, miR-199 and miR-214 are upregulated with hypertrophy.
Heart failure remains a main cause of morbidity and mortality, it is a progressive disorder initiated by myocardial injury and is characterised by fibrosis, hypertrophy and cardiac dysfunction. A study by Thum et.al.(2009), showed the observation of a link between miRNA expression and heart failure. They used a mouse model and observed that miR-21 was the most significantly altered miRNA, being strongly increased in heart failure. They then went on to investigating a potential role for increased miR-21 expression in heart failure and the in vitro findings showed a synthetic miR-21 antagonist increased the percentage of apoptotic fibroblasts, whereas miR-21 overexpression decreased it.
1.6.1 miRNA and radiotherapy
miRNAs regulate gene expression, therefore it is likely that miRNAs regulate genes involved in the cellular response to potenially lethal stressors. Ionising radiation used in the treatment of malignancy, can cause cellular damage and stress. Studies have shown an effect of radiation on miRNA expression patterns both in vitro and in vivo. Simone N.et.al.(2007) carried out a miRNA microarray analysis which showed that irradiation alters the expression of miRNA species. Expression levels in 10 of the 17 miRNA species increased after exposure to radiation. miRNA expression for let-7a and let7b were validated by RT-PCR. Expression of both let-7a and let7b decreased significantly after treatment with radiation. They then went on to determine whether changes in miRNA expression was related to radiation dose. The results showed Dose dependent, linear decrease in miRNA expression from 0.25 to 1 Gy one hour after irradiation. No further dose dependent decrease was observed in the higher dose ranges. This show that miRNA expression does change with radiation dose. 1Gy may produce the maximum alteration in let-7a and let-7b Simone N.et.al.(2007).
1.6.1 Radiotherapy and cardiac damage the miRNA connection?
Currently no studies have been carried out to determine whether radiation-induced long-term changes in miRNA profiles can provide a link between radiotherapy, biological ageing and an increased risk of heart disease. In this project, an initial literature search will be carried out to assess current understanding of links between irradiation, miRNA profiles, cardiac disease, biological aging and biomarkers in peripheral blood lymphocytes. RNA of sufficient quantity and quality will be extracted and testing will be carried out by qPCR of candidate miRNAs known to be altered following irradiation and those associated with heart disease. Observations will be made to see if radiotherapy alters the expression profile of miRNAs extracted from these samples and if there is a positive outcome, it will allow further validation in an in vivo model to ascertain whether changes observed in blood relate to those observed in cardiac tissue by comparing miRNA profiles on blood and heart tissue. This study will lead to the hypothesis of can alterations in specific miRNA's following radiotherapy predict the likelihood of developing cardiac disease.
1.7 Aims of study
The hypothesis of this project is short and long term radiotherapy induced complications can be predicted with genotyping and in vitro markers, pre and post radiotherapy. The aims of this study is to measure baseline in vitro biological ageing assays, such as apoptotic response to irradiation and DNA damage and repair kinetics in peripheral blood lymphocytes. To develop testing of telangiectasiae predisposing genetic changes uses Single Nucleotide Polymorphisms (SNP) assays and to finally determine whether radiation-induced long-term changes in miRNA profiles can provide a mechanistic link between radiotherapy, biological ageing and an increased risk of heart disease.