EGFR belongs to a family of four receptors including the EGFR, ERBB2, HER3, and HER4 receptors. The EGFR gene is located on 7p11.2 and produces a glycoprotein with a molecular weight of 170kDa with high affinity for the EGF ligand and is capable of mediating multiple signal transduction pathways
All T-cells originate from haemopoietic stem cells in the bone marrow. Haemopoietic progenitors in the Thymus expand and divide in the thymus resulting in a large population of immature thymocytes. Approximately 2% of thymocytes survive development in the thymus and are released to become immunocompetent T cells. Contribution of the thymus to T cell generation decreases with age in correlation with shrinkage of the thymus (3% per year at middle age) giving a corresponding decrease in thymic production of naïve T cells.
T Lymphocytes play a central role in cell mediated immunity and are distinguishable from other lymphocytes, including B cell, on account of the presence of the T cell receptor present on the cell surface. There are several subsets of T cells including; Cytotoxic CD8+ T cells (Tc), central memory T cells(Tcm), effector memory T cells (Tem), naturally occurring CD4+ regulatory T cells (Treg), adaptive Treg cells (Th1/Th3), Natural Killer cells (NK) and low incidence γδ (gamma, Delta) T cells.
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EGFR is a known self- antigen [Schuler et al 2011], therefore a mediated immune response in the negative control samples may have caused the release of cytotoxic T cells targeting the EGFR gene resulting in a reduction in the number of signals observed. This may explain why some of the cells quantified demonstrated no EGFR signals. The immune response may have originated from the donors due to unknown inflammation or fever that would not have been disclosed before donation. Another possibility is that the DNA isolated and extracted from the RP5-1091E12 PAC was unsuccessful therefore the probe generated would not correctly label the EGFR gene . This is unlikely as the same source of isolated PAC DNA was used for each FISH preparation and had been successful at various points throughout the study, however it is possible, though unlikely, that the DNA may have been contaminated during the study.
As well as its association with various cancers EGFR amplification has also been associated with features of bronchial asthma in relation to epithelial damage and airway remodelling. Studies performed in 2000,[Puddicombe et al 2000] showed that biopsies from asthmatic subjects displayed strong EGFR immunostaining in areas of epithelial damage as well as high EGFR expression in asthmatic epithelia that remained intact. It was later disclosed that the female negative donor used in this study was in fact a chronic asthmatic, which may account for the anomalous results indicative of EGFR amplification, however it is unconfirmed whether EGFR amplification relating to asthma can be observed in blood lymphocytes. Another possibility is that the negative control samples did in fact come from donors that have gained extra EGFR copies due to the presence of a cancer of which they are not aware, again this is unconfirmed.
Artefacts of unknown origin present in the slides were likely due to mechanical error during slide preparation, or, could indeed be attributed to the use of lymphocytes as a study model. Lymphocytes travelling through the bloodstream could acquire any manner of artefacts whilst travelling the donor's body, this is unconfirmed and without further study it is difficult to determine how the artefacts arose and from where they originated. As well as being present in random positions on the slides viewed artefacts also occurred positioned within the interphase spreads which may have masked the EGFR signals, also a contributing factor to the reduced number of normal signals present.
As expected the results from the MDA-MB-468 samples showed significant amplified EGFR signals with no normal signals recorded. Gene amplification associated with Cancer is understood to be largely associated with increased transcription of (proto) oncogenes, however, whether or not this is the dominant initiating factor and promoter of the progression of cancer is still to be determined. Overexpression of EGFR may undergo regulation at a transcriptional level, however, it is also known to be, in many cases, largely mediated by chromosomal alterations resulting in an increase in copy number. Further studies investigating this matter are fairly limited due to the fact that there are few models that retain the conserved EGFR signal once the tumour cells are cultured, making MDA-MB-468 a useful and fairly widely used model for studying EGFR signals.
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As mentioned previously amplification of the EGFR gene is largely associated with the BFB cycle which results in daughter cells with genes with different copy numbers. It is thought that initiation of the BFB cycle occurs when fragile sites distal to the gene of interest undergo chromosomal breakage, which results in asymmetric segregation during mitosis and therefore non- homologous daughter cells. FRA 7 is a known fragile site located on 7p11.2, enclosing the EGFR gene at its centre and it is thought that chromosomal damage to the FRA 7 site distal to the EGFR gene is the initiating factor for the BFB cycles resulting in amplification of the EGFR gene associated with Cancer. Amplifications of the EGFR gene can be detected in vivo sometimes as double minutes but in most cases homologous staining regions in ladder like amplification structures can be observed [Agelopoulous et al 2010].
Due to an inability to obtain the blood from breast cancer patients for comparative analysis, as mentioned previously, it was not possible to meet the original aim of this study. The 2nd aim of the study was met giving results indicative of the use of T lymphocytes as a model for studying the EFGR gene. However, it is not possible to give a definitive outcome regarding the use of lymphocytes as a study model due to the small sample size and anomalous results. Further studies are required, using a larger sampling pool, with a more rigid screening process for the donors used, to rule out any other factors that may have biased the results in some manner e.g. non- smokers, no illness for 6 months and disclosure of health problems that may affect the results. Also, with less time constraints it would have been ideal to run the isolated DNA through electrophoresis to confirm that the correct DNA had been isolated.
If further investigations were to corroborate the results from this study, it would indicate that along with EGFR's self -antigenic properties, lymphocytes are too unpredictable a model with too little significant and consistent results to use as a model for studying the EGFR gene effectively at this time.