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Breast cancer is the most common form of cancer in women, and in the UK affects one in nine women with almost 44,000 annual cases, resulting in 12,000 mortalities (Evans D.et.al.2007). At present the treatments available for breast cancer patients include; surgery (lumpectomy and mastectomy), chemotherapy, hormonal therapy, targeted therapy and radiotherapy. As age increases, the likelihood of developing breast cancer also increases, possibly resulting from unrepaired genetic damage that incurred during the course of an individuals' life. (Ronckers C.et.al.2005). The correlation between age and reported breast cancer cases is shown in figure 1.1.
Figure 1.1: Breast Cancer occurrence correlated with age
The number of reported breast cancer cases increases with age, with the 60-64 age group identified as most at risk. Graph adapted from : (http://info.cancerresearchuk.org/cancerstats/types/breast/incidence/)
Breast cancer mortality has fallen in the last decade due to breast cancer screening and improvements to the way breast cancer is treated. Early detection, successful treatment and predication of complications is essential for the treatment of breast cancer ONS (2007)
Breast cancer and chemotherapy
Chemotherapy is a systemic therapy used to treat all stages of breast cancer. It works by destroying cancer cells by disrupting their DNA, protein production and also preventing cell division. Not all breast cancer patients are at the same stage of cancer and therefore the type and dose of drug given is individualised specifically for their requirements. The dose given is very important as it majorly determines the anti-tumour activity and also the toxicity of the chemotherapeutic agent. The route of administration is either intravenously (through the vein) or orally (tablets). A complete course of chemotherapy lasts 4-6 months. Polychemotherapy, a chemotherapy regime involving the use of more than one drug, is regarded as being more effective than using a single chemotherapeutic agent. Figure 1.2a shows that for the age group less than 50 years, the recurrence of cancer was marginally lower with the use of polychemotherapy. Figure 1.2b shows that breast cancer mortality was lower with the use of polychemotherapy.
Figure 1.2a and 1.2b: Breast Cancer recurrence and mortality rate after polychemotherapy
Radiotherapy uses high energy x-rays to destroy DNA. An advantage of this therapy is that it is a focused treatment where the x-rays are aimed directly at the site of the tumour and although normal cells can also be damaged, they can often repair themselves. There are two ways of applying radiotherapy; internally (bracytherapy) or externally.(Violet.et.al.2004). To aid the planning of a breast cancer patients treatment, they are generally categorized according to clinical criteria, this includes the stage and grade of the tumour. In radiotherapy, external tangential beams are aimed at the tumour site post-conservation surgery, or to the chest wall after mastectomy (Griffiths and short 1994).
The initial advantage of radiotherapy on a patient's long term survival often results in complications. For instance patients may undergo a series of normal tissue reactions, and tissue toxicity may result in asymptomatic alteration in the function and structure of the tissue. (Bentzen et.al.2003). The key principles of radiotherapy include; the ability to achieve high radiation dose into the tumour, minimize dose into surrounding normal tissue and to avoid adverse complications.
Figure 1.3: Percentage of overall breast cancer survival post radiotherapy
The percentage of overall breast cancer survival after radiotherapy treatment in 1974 was 49% and by 1999 this had increased to 90%, demonstrating that radiotherapy regime has improved. Graph adapted from Survival of invasive breast cancer according to NPI (Eur J Cancer: Blamey et al 2007).
Radiotherapy and DNA damage
Photons and electrons in the radiation source causes ionisation of water molecules producing free radicals (OH radicals). This ionises the atom present in the DNA molecule. In response to DNA damage, cells activate a signaling cascade known as the DNA damage response (DDR) the main function of which is to promote the repair of the damage while delaying cell cycle progression until repair can be completed. DNA damage can be repaired through several mechanisms including; the direct chemical reversal of the damage, base excision repair, nucleotide excision repair and mismatch repair. Mention NHEJ and homologous recombination. If DNA repair is unsuccessful- apoptosis is initiated, however defective apoptosis mechanism can result in cell survival with excess DNA damage and the uncontrollable replication of cells with DNA damage can result in carcinogenesis, reference here.
Adverse effects of radiotherapy
Radiotherapy has improved breast cancer survival as shown in figure 1.3, but some patients go on to develop complications, which are either local (skin and subcutaneous) or more serious complications such as the increased risk of developing secondary primary malignancies. Toxicity experienced from radiotherapy can be separated into two distinct groups: (i) early/acute which occur within 90 days of undergoing treatments, and (ii) late reactions occuring at any time period after this for upto a year. (Van der Kogel 1993). Acute adverse effects from exposure to radiation are a result of cell death, which may be due to the cells failure to pass through mitosis following irradiation. Such effects include erythema, which is redness of the skin caused by capillary congestion. Acute reactions are not permanent and recovery is generally rapid. Late adverse effects arise from damage of blood vessels and connective tissue cells. Such effects include telangiectasia, which is surfacing of dilated blood vessels and lymphedema, a condition of localised fluid retention and tissue swelling. Late effects are considered to be more permanent and are a preventive factor in radiotherapy dosage.
Cardiac damage after radiotherapy
Current methods of breast cancer radiotherapy may involve irradiation of the heart due to the physical location of the tumour, such treatments result in a 27% increase in mortality from heart disease and possibly attenuating the benefits of radiotherapy on overall survival, refer to figure 1.4. Women with left sided breast cancer are more likely to develop cardiovascular disease, this is due to the location of heart. When radiotherapy is assigned to both left and right sided breast lesions, at least 1Gy dose from scattered irradiation is given to the heart (Li.et.al.2000). The left anterior descending artery is most likely to be affected, due to its anatomical location close to the radiation field, as shown in figure 1.3, and multiple studies have demonstrated fixed and reversible cardiac perfusion defects. (reff).
Many radiation induced cardiac problems results from both microvascular and macrovascular damage to the heart. The damage to the microvascular component results from endothelial cell damage within the heart leading to ischeamia and progressive obstruction of the vessel lumen. An additional adverse effect of radiotherapy is premature coronary artery disease, in which one or more of the blood vessels is clogged with plaque, this causes the heart muscle not to receive enough oxygen and nutrition to function properly and results in chest pain (angina). If the blood vessel is totally blocked, it can result in a heart attack.
Figure 1.3: Breast cancer radiotherapy involving irradiation to the heart
In radiotherapy a number of tangential beams are directed towards the tumour-originating tissue after conservation surgery or to the chest wall after mastectomy.
Figure 1.4: to get a proper table from paper ask Julian and then write here
Markers of cardiac damage
Can long-term radiotherapy damage be predicted with new biomarkers?
Adverse reactions to radiotherapy are a major clinical problem and often difficult to predict. Cancer patients react differently with respect to their normal tissue response to radiotherapy. Taking this into account during treatment planning of the patient could enable therapeutic strategies to be individualised. Hence, predicting a patients response to radiotherapy is at present a sought after goal. In the past however, studies have concentrated on designing assays which consider cellular radiosensitivity or sub-celullar damage end-points. In spite of this, current studies are focusing on the notion that normal tissue radiosensitivity could be predicted from individual genetic profiles.
Present day technological advances are being used to uncover the molecular basis of normal tussue complications, and impart a list of radiosensitivity candidate genes. Micro-array based gene expression profiling can clarify expression before and after irradiation of cells or tissues and new radiation induced genes could be recognised. (Andreassen C 2005).
Ataxia Telangiectasia mutated gene (ATM) and Ataxia Telangiectasia
Cutaneous telangiectasia is one of the common late normal tissue effects following radiotherapy and can arise from six months to many years after radiotherapy treatment. Telangiectasia is caused by vascular damage in small dilated blood vessels in the skin and appears as flattened red marks on the skin and mucous membranes (see figure 6) and is often seen in normal individuals where it is associated with aging or in response to excessive alcohol use or sun exposure. A number of studies have attempted to discover genetic variants that are associated with telangiectasia in patients post radiotherapy treatment. (Giotopoulous et.al.2009) identified a polymorphism (T399Q) in the XRCCI gene which is involved in DNA repair and this polymorphism was linked with an increasing risk of developing telangiectasia. (which pathway and give stats….read paper)
Figure 6: A clinical view of dilated blood vessels typical of telangiectasia
As telangiectasia can be unsightly, it is primarily considered as a cosmetic problem; however a recent study by Tanteles G.et.al.2009 presented evidence that women who developed telangiectasia within years following breast cancer radiotherapy were more likely to develop radiation induced cardiovascular disease. (WHAT are the stats). These data suggest that telangiectasia may be considered as a useful marker of prospective radiation-induced cardiac complications.
The human Ataxia telangiectasia mutated (ATM) gene is located at position 11q22-q23 and encodes for proteins such as?? with important function in the recognition of DNA damage and the activation of pathways resulting in cell cycle arrest, DNA repair or apoptosis (Kastan MB et.al.2000). The ATM gene is mutated in rare cancer prone individuals with a heritable form of ataxia-telangiectasia, which is primarily caused by an immunodeficiency which is??? Predisposing AT patients to cancer. Whilst treating AT patients malignancies with radiotherapy (Gotoff et.al.1967) noticed that the patients were hypersensitive to the effects of irradiation. It was proposed that this hypersensitive response was as a result of impaired ATM protein function and inefficient DNA damage repair.
First author, year [Ref]
DNA damage repair
Risk of subcutaneous fibrosis significantly associated with the codon 1883 Asp/Asn and Asn/Asn genotypes.
Acute and late adverse reactions
DNA damage repair
No ATM mutations detected in 23 patients with severe acute or late toxicity.
Acute and late adverse reactions
DNA damage repair
No indications of increased acute or late radiosensitivity in 10 patients being heterozygous for pathogenic ATM mutations.
Late: moderate to severe side effects
DNA damage repair
Significant associations between SNPs and pleural thickening and lung fibrosis.
Late: severe side effects
DNA damage repair
Sequence variants, in particular 5557 G> A, may be associated with late adverse reactions.
Acute and late adverse reactions
DNA damage repair
Significant association between missense variants and severe subcutaneous late damage.
Table 2: A better table name?!?!!?!?
Biological Ageing, breast cancer and radiotherapy
Biological ageing is due to the accumulation of DNA damage predominantly due to impaired repair activity leading to mutations and resulting in cancer. The rate of biological ageing is shown by both environmental and genetic factors. In a study by Docherty.et.al.2007 it was observed that in vitro apoptotic response in normal circumstances in peripheral blood lymphocytes was 0.5% per year, however in breast cancer patients who had been exposed to ionising radiation it fell by 15%, one year post-radiotherapy, which equates to 30 years of biological aging over a one year period post irradiation. Talk about biological ageing syndrome: refer to julians email.
The cellular response to DNA damage is initiated by the recognition of injured DNA, by damage assessment enforced by checkpoints. Apoptosis is a mechanism for eliminating cells with DNA damage. Defects in apoptotic pathways have been proven to play a role in 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 membranes. The cells also shrink, and the membranes begin to bleb and form membrane-enclosed vesicles.
This pathway is induced by stress signals comprising of; hypoxia, DNA damage and loss of survival signals. (Zhang et.al.2005).
This pathway is induced by the tumour necorsis factor family, which is imprtant in T lymphocyte development and function Hajra et.al.2004).
This pathway is induced in neuronal tissue
Table 3: The three different mechanisms by which a cell undergoes apoptosis
Radiation-induced Apoptotic response
Irradiation-induced apoptosis is a result of DNA double strand breaks (Steel 2002). Such double strand breaks can activate ATM, DNA dependent, protein kinase mediated by p53 activation by either direct phosphorylation at the N-terminal serine 15. This leads to transcriptional activation of p21 protein which causes cell cycle arrest at the G1 checkpoint (Lavin and Khanna 1999). Many studies have proven that apoptotic response to DNA damage decreases with aging. In a study carried out by (Polyak et.al.1997), there was a reduction in apoptotic response in lymphocytes exposed to a 5Gy radiation in mice with increasing age.
Sub-G1 peak assay
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 (Camplejohn et.al.2005). In apoptosis, the DNA fragments and forms a peak below that seen in the G1 phase of the cell cycle, this is measured by assessing propidium iodide concentrations that bind to DNA. In regards to a DNA histogram, the cell population observed in a peak corresponding to low DNA levels (Haploid cells) is a representative of the number of cells at the G0/G1 checkpoint. The cell population observed in the second peak corresponding to a larger DNA content (Diploid cells) is a representative of the number of cells at the G2/M checkpoint in the cell cycle. Cells in the s phase will be shown between the haploid and diploid cell peaks.
The alkaline single-cell microgel electrophoresis (Comet) assay is a method used to detect DNA damage and repair, involving the encapsulation of cells in agarose suspension, lyses of cells in neutral or alkaline conditions, and electrophoresis of the suspended lysed cells. This is followed by means of 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. Electrophoresis causes the damaged DNA to migrate away from the undamaged DNA forming a 'comet', in which the 'head' of the comet is the undamaged DNA and the 'tail' contains fragmented DNA. Therefore, the more damaged DNA there is within a cell, the greater the volume of DNA within the comet tail. In a study by (Vrhovac V.et.al.2003) the alkaline comet assay was used as a biomarker to monitor the effect of ongoing exposure to ionising radiation in 50 medical workers, and 50 unexposed control individuals using peripheral blood leukocytes. The results of this study clearly demonstrated that the levels of DNA damage observed in irradiated individuals was much higher than those in the control groups and this confirms that the comet assay is a useful marker to assess the impact of radiation exposure on DNA damage.
Figure 1.5: COME OUT WITH GOOD LABEL NAME
miRNAs and cardiac damage
miRNAs are short, non coding RNA molecules of approximately 21 bases in length, which can regulate gene expression via the inhibition of messenger RNA (mRNA) translation, or mRNA degradation. (reference) It is this regulation and deregulation that influences the development of normal cells into disease cells, as they have been implicated in many cellular pathways including development and are associated with alterations in phenotype and disease including cancer and cardiac problems. Understanding how miRNA influences the development of normal cells into disease cells will lead to better diagnosis and treatment for cardiac disease.
The involvement of miRNAs in cardiac 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. Certain microRNAs were up regulated and down regulated in mice subjected to thoracic aortic banding. These miRNAs were also observed to be deregulated in failing human hearts, suggesting they determined a diagnostic molecular signature for cardiac pathogenesis. This shows that miRNAs are implicated in cardiac function. (Rooij.E.et.al.2006). In 2008, Rooij.E.et.al demonstrated that miR-1, miR-229,miR-133 and miR-150 were found to be downregulated during pathological cardiac remodeling, whereas miR-21, miR-23a,miR-125, miR-195,miR-199 and miR-214 are up regulated with hypertrophy. Heart failure remains a main cause of morbidity and mortaility, it is a progressive disorder initiated by myocardial injury and is characterized by fibrosis, hypertrophy and cardiac dysfunction.
A study by (Thum et.al.2009), further suggests a relationship between the alterations in miRNA expression profiles and cardiac failure. Significant alterations were observed in the levels of miR-21. Further work by this group has shown that in vitro that the introduction of a synthetic miR-21 antagonist increased the percentage of apoptotic fibroblasts, whereas over-expression of miR-21 decreased them.
miRNA and radiotherapy
Radiotherapy and cardiac damage the miRNA connection
Aims of study
In this study an attempt will be made to:
Predict late normal-tissue reactions by genotyping single nucleotide polymorphisms (SNPs) in the ATM gene of 634 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 undergoing radiotherapy, immediately after treatment, and six weeks post completion of radiotherapy. This will be measured in vivo with cardiac magnetic resonance imaging (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.